Cells and method of cell culture

ABSTRACT

The invention relates to a method of cell culture where the cells are modified to reduce the level of synthesis of growth and/or productivity inhibitors by the cell. The invention also relates to a method of cell culture for improving cell growth and productivity, in particular in fed-batch culture of mammalian cells at high cell density. The invention further relates to a method of producing cells with improved cell growth and/or productivity in cell culture and to cells obtained or obtainable by such methods.

This application is a divisional of U.S. application Ser. No. 15/762,345filed Mar. 22, 2018, which is a § 371 filing of PCT/IB2016/055666 filedSep. 22, 2016, which claims the benefit of priority to United StatesProvisional Application Nos. 62/222,555 filed Sep. 23, 2015 and62/396,475 filed Sep. 19, 2016; the entire contents of which areincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application was filed electronically via EFS-Web and includes anelectronically submitted sequence listing in .txt format. The .txt filecontains a sequence listing entitled “PC72254B_SeqListing_ST25.txt”created on Jun. 18, 2020 and having a size of 2 KB. The sequence listingcontained in this .txt file is part of the specification and is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to a method of cell culture where the cells aremodified to reduce the level of synthesis of growth and/or productivityinhibitors by the cell. The invention also relates to a method of cellculture for improving cell growth and productivity, in particular infed-batch culture of mammalian cells at high cell density. The inventionfurther relates to a method of producing cells with improved cell growthand/or productivity in cell culture and to cells obtained or obtainableby such methods.

BACKGROUND OF THE INVENTION

Proteins have become increasingly important as diagnostic andtherapeutic agents. In most cases, proteins for commercial applicationsare produced in cell culture, from cells that have been engineeredand/or selected to produce unusually high levels of a particular proteinof interest. Optimization of cell culture conditions is important forsuccessful commercial production of proteins. Mammalian cells haveinefficient metabolism which causes them to consume large amounts ofnutrients and convert a significant amount of them to byproducts. Thebyproducts are released into the culture and accumulate over the courseof the culture. Lactate and ammonia, known to be the conventionalinhibitors of cells in culture, are the two major byproducts of cellularmetabolism that accumulate to high levels in culture and beyond certainconcentrations, they start inhibiting the growth and productivity ofcells in culture. Cell culture methods aimed at reducing the amount oflactate and ammonia in the cell culture medium have been developed andcan increase the growth and the productivity of mammalian cells. Thecell growth, however, still slows down even when concentrations oflactate and ammonia are kept low, thereby limiting the maximum celldensity and productivity of the cells.

Therefore, there is a need for the development of improved cell culturesystems for optimum production of proteins. In particular there is aneed for cell culture methods providing an increased viable cell densityand/or titer and in particular for cells which are modified to reducethe level of synthesis of growth and/or productivity inhibitors.

SUMMARY OF THE INVENTION

The invention relates to a method of cell culture comprising (i)providing cells in a cell culture medium to start a cell cultureprocess, wherein the cells are modified to reduce the level of synthesisof growth and/or productivity inhibitors by the cell.

The invention also relates to method of producing cells with improvedcell growth and/or productivity in cell culture comprising the steps of:

-   -   (i) identification of a cell metabolite or metabolites which are        cell growth and/or productivity inhibitors,    -   (ii) identification of the cell metabolic pathway or pathways        resulting in the synthesis of the cell growth and/or        productivity inhibitors,    -   (iii) identification of one or more genes in the cell metabolic        pathway or pathways encoding an enzyme which catalyses the        synthesis of the cell growth and/or productivity inhibitors or        metabolic intermediates thereof, and/or one or more genes        encoding an enzyme in a metabolic pathway branching or directly        branching therefrom,    -   (iv) modifying the expression of the one or more genes to reduce        the level of synthesis of the cell growth and/or productivity        inhibitors.

The invention further relates to a cell comprising one or more modifiedgenes which reduces the level of synthesis of growth and/or productivityinhibitors by the cell, in particular wherein the one or more modifiedgenes is selected from Bcat1, Bcat2, Auh, Mccc1, Mccc2, Ivd, PCDB1,QDPR, Hpd, Hgd and Pah, wherein the modification increases or decreasesthe gene expression, and the use of the foregoing cells for theexpression of a recombinant protein or polypeptide.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the growth characteristics and metabolic profiles of CHOcells in conventional fed-batch process. Reported data includes viablecell densities (closed squares), culture lactate levels (closedtriangles) and ammonia levels (closed diamonds). Also, reported is theharvest titer (day 12 protein concentration).

FIG. 1B shows the growth characteristics and metabolic profiles of CHOcells in a HiPDOG process. Reported data includes viable cell densities(closed squares), culture lactate levels (closed triangles) and ammonialevels (closed diamonds). Also, reported is the harvest titer (day 12protein concentration).

FIGS. 2A, 2B, and 2C show graphs indicating the viable cell density ofGS-CHO cells (hereafter GS-CHO, Cell line A) at day 0, Day 3 and Day 6when exposed to increasing concentrations of 2-hydroxybutyric acid (FIG.2A), homocysteine (FIG. 2B) or indolecarboxylic acid (FIG. 2C). GS-CHOcells were inoculated in Medium A at low viable cell densities and weretreated with reported concentrations of the inhibitors, individually.The effect of the inhibitors on growth of the cells was monitored for 6days.

FIGS. 3A and 3B show the effect of increasing concentrations of twometabolites, 4-hydroxyphenylpyruvate (FIG. 3A) and phenyllactate (FIG.3B) on viable cell density of the GS-CHO cells. GS-CHO cells wereinoculated in Medium A at low viable cell densities and were treatedwith reported concentrations of the inhibitors, individually.

FIGS. 4A and 4B, and FIGS. 5A and 5B show the effect of increasingconcentrations of four metabolites, indolelactate (FIG. 4A),3-(4-hydroxyphenyl)lactate (FIG. 4B), sodium formate (FIG. 5A) andisovalerate (FIG. 5B), on viable cell density of the GS-CHO cell. GS-CHOcells were inoculated in Medium A at low viable cell densities and weretreated with reported concentrations of the inhibitors, individually.

FIG. 6 shows the effect of four metabolites (4-hydroxyphenylpyruvate,indolelactate, phenyllactate, and 3-(4-hydroxyphenyl)lactate) on viablecell density of GS-CHO cells as compared to a control where cells arecultured in the absence of the metabolites. GS-CHO cells were inoculatedin Medium A at low viable cell densities and were treated with freshmedia alone or fresh media comprising the above mentioned fourmetabolites in combination at concentrations that were detected on day 7of the HiPDOG culture (See Table 4). The effect of the inhibitors ongrowth of the cells was monitored for 6 days.

FIG. 7A shows the viable cell densities of GS-CHO cells in ‘HiPDOG’process (closed squares) and ‘low AA+HiPDOG’ (closed diamonds) process.

FIG. 7B shows the culture titer (IgG) at different days in ‘HiPDOG’process (closed squares) and ‘low AA+HiPDOG’ (closed diamonds) process.

FIGS. 8A and 8B, and FIGS. 9A and 9B show the amino acid concentrationsof four amino acids in ‘amino acid restricted HiPDOG process (LowAA+HiPDOG)’ (closed squares) and the ‘HiPDOG’ process (closed diamonds).The four amino acids include tyrosine (FIG. 7A), tryptophan (FIG. 8B),phenylalanine (FIG. 9A), and methionine (FIG. 9B).

FIG. 10A shows the viable cell densities of GS-CHO cells during a cellculture process using different conditions disclosed in Example 5(HiPDOG1 (closed squares), HiPDOG2 (closed circles), Low 4AA+HiPDOG(closed diamonds), Low 8AA+HiPDOG (closed triangles)).

FIG. 10B shows the culture titer (IgG) at different days in a cellculture process using GS-CHO cells and different cell culture conditionsdisclosed in Example 5 ((HiPDOG1 (closed squares), HiPDOG2 (closedcircles), Low 4AA+HipDOG (closed diamonds), Low 8AA+HipDOG (closedtriangles)).

FIGS. 11A and 11B, FIGS. 12A and 12B, FIGS. 13A and 13B, and FIGS. 14Aand 14B show the concentrations of tyrosine (FIG. 11A), methionine (FIG.11B), phenylalanine (FIG. 12A), tryptophan (FIG. 12B), leucine (FIG.13A), threonine (FIG. 13B), glycine (FIG. 14A) and serine (FIG. 14B)during the cell culture of GS-CHO cells using conditions disclosed inExample 5 ((HiPDOG1 (closed squares), (HiPDOG2 (closed circles), Low4AA+HipDOG (closed diamonds), Low 8AA+HipDOG (closed triangles)).

FIGS. 15A and 15B, and FIG. 16 show the concentration of3-(4-hydroxyphenyl)lactate (FIG. 15A), isovalerate (FIG. 15B) andindole-3-lactate (FIG. 16 ) at day 5, day 7 and day 9 of the cellculture of GS-CHO cells using conditions disclosed in Example 5((HiPDOG1 (closed squares), HiPDOG2 (closed circles), Low 4AA+HipDOG(closed diamonds), Low 8AA+HipDOG (closed triangles)).

FIGS. 17A and 17B shows the viable cell densities of GS-CHO cells andculture titer (IgG) during a cell culture process using conditionsdisclosed in Example 5 ((Low 4AA+HiPDOG (closed diamonds), Low8AA+HipDOG (closed triangles)).

FIGS. 18A and 18B show the viable cell densities of GS-CHO cells andculture titer (IgG) during a cell culture process using conditionsdisclosed in Example 6 ((HiPDOG (closed squares), Low 8AA+HipDOG (closedtriangles)).

FIGS. 19A and 19B, and FIG. 20 show the concentration of3-(4-hydroxyphenyl)lactate (FIG. 19A), isovalerate (FIG. 19B) andindole-3-lactate (FIG. 20 ) at day 5, day 7 and day 10 of the cellculture of GS-CHO cells using conditions disclosed in Example 6 ((HiPDOG(closed squares), Low 8AA+HipDOG (closed triangles)).

FIGS. 21A and 21B show the viable cell densities of GS-CHO cells andculture titer (IgG) during a cell culture process using differentconditions disclosed in Example 6 ((HiPDOG (closed squares), Low4AA+HipDOG (closed diamonds)).

FIGS. 22A and 22B, and FIG. 23 show the concentration of3-(4-hydroxyphenyl)lactate (FIG. 22A), isovalerate (FIG. 22B) andindole-3-lactate (FIG. 23 ) at day 5 and day 7 of the cell culture ofGS-CHO cells using conditions disclosed in Example 6 ((HiPDOG (closedsquares), Low 4AA+HipDOG (closed diamonds)).

FIGS. 24 and 25 respectively show the phenylalanine/tyrosine and theleucine pathways including metabolic enzymes involved in the pathway andits branches, also tabulated is RT qPCR measure of transcript abundanceindicated for the genes of the pathway as cycle number or C_(T)(Threshold Cycle). The C_(T) values of the metabolic genes are tabulatedand compared to the C_(T) value of a well characterized housekeepinggene, Beta Actin. The difference between the C_(T) of the target geneand the C_(T) of Beta Actin is tabulated as the ΔC_(T). High ΔC_(T)value indicates low gene expression level.

FIG. 26 : Gene expression analysis of phenylalanine/tyrosine pathwaygenes using RT-qPCR assay. Expression levels of phenylalanine/tyrosinepathway genes in CHO cell line A and CHO parental cell line. Data isplotted as log of the ration of gene of interest transcript level toB-Actin transcript level. Higher value indicates higher expression ofthe gene. Schematic of phenylalanine/tyrosine catabolic pathway.Phenylpyruvate, phenyllactate, 4-hydroxyphenylpyruvate and3-(4-hydroxyphenyl)lactate are inhibitory intermediates and byproductsof the pathway. Enzymes in light grey shade are those which areexpressed at very low levels.

FIGS. 27A and 27B: Gene expression analysis of tetrahydrobiopterin (BH₄)biosynthesis and regeneration pathway genes using RT-qPCR assay. (A)Schematic of tetrahydrobiopterin (BH₄) biosynthesis and regenerationpathway. GTP is the carbon source for biosynthesis of BH₄ catalyzed bythree enzymes GCH1, PTS and SPR. BH₄ regeneration pathway consists oftwo enzymes, PCBD1 and QDPR, that together regenerate BH₄ fromBH₄-4a-carbinolamine, which is the product of the reaction catalyzed byPAH. (B) Expression levels of tetrahydrobiopterin (BH₄) biosynthesis andregeneration pathway genes in cell pools of CHO cell line A, CHO cellline B or CHO parental cell line overexpressing mouse PAH. Data isplotted as log of the ration of gene of interest transcript level toB-Actin transcript level. Higher value indicates higher expression ofgene.

FIGS. 28A, 28B, 28C, and 28D: Plasmids used for generating transgenicCHO cell lines expressing mouse PAH, HPD, HGD, and PCBD1. (A) Plasmidmap of the expression vector for mouse PAH. (B) Plasmid map of theexpression vector for mouse HPD. (C) Plasmid map of the expressionvector for mouse HGD. (D) Plasmid map of the expression vector for mousePCBD1.

FIGS. 29A and 29B: Growth kinetics of quadruple transfected (4x-tfxn) ornegative quadruple transfection control (4x-control-tfxn) cell pools intwo subsequent tyrosine-free cell culture passages. Length of the firstpassage was three days and the length of the second passage was fivedays. (A) Cell pools derived from CHO cell line B. (B) Cell poolsderived from CHO parental cell line. ▴: 4x-tfxn, ▪: 4x-control-tfxn.

FIGS. 30A, 30B, 30C, and 30D: Comparison of the performance of cell lineB derived quadruple transfected (4x-tfxn) cell pools in tyrosine-freeHiPDOG fed-batch process as compared to the negative quadrupletransfection control (4x-control-tfxn) cell pools in original HiPDOGfed-batch process (tyrosine supplemented). (A) Viable cell density (B)Viability (C) Titer (D) Levels of 3-(4-hydroxyphenyl)lactate ♦: 4x-tfxn,▪: 4x-control-tfxn.

FIGS. 31A and 31B: Effect of different concentrations of2-methylbutyrate (A) or isobutyrate (B) on growth of CHO cell line A

FIGS. 32A, 32B, 32C, and 32D: Suppressing the accumulation of2-methylbutyrate (B) and isobutyrate (D) by controlling theconcentration of isoleucine (A) and valine (C) at low levels in thefed-batch cultures of CHO cell line C. ▴: Low AA, ♦: Low AA, ▪: Control

FIGS. 33A and 33B: Gene expression analysis of leucine pathway genesusing RT-qPCR assay. (A) Expression levels of leucine pathway genes inCHO cell line A and CHO parental cell line. Data is plotted as log ofthe ration of gene of interest transcript level to B-Actin transcriptlevel. Higher value indicates higher expression of gene. (B) Schematicof leucine, isoleucine and valine catabolic pathways. Isovalerate,2-methylbutyrate and isobutyrate are growth inhibitory byproducts ofleucine, isoleucine and valine catabolic pathways, respectively. BCAT1(BCAT2) and BCKHDA/BCKHDB are shared by all the three catabolicpathways. Knockdown or knockout or inhibition, for example inhibitionwith a small molecule inhibitor molecule, of enzyme activity of BCAT1(and/or BCAT2), or BCKDHA/BCKDHB will concurrently reduce thebiosynthesis of the three inhibitory metabolites (isovalerate,2-methylbutyrate and isobutyrate).

FIGS. 34A, 34B, 34C, and 34D: Western blot for probing the levels ofBCAT1 and B-Actin in CHO cells transiently transfected independentlywith five miRNAs against BCAT1. (A) B-Actin protein levels inuntransfected or transfected CHO parental cell line (B) BCAT1 proteinlevels in untransfected or transfected CHO parental cell line (C)B-Actin protein levels in untransfected or transfected CHO cell line B(D) BCAT1 protein levels in untransfected or transfected CHO cell lineB. Gel loading details for all the gels are as follows. Lane 1: proteinladder, lane 2: untrasfected cells, lane 3: cells transfected with BCAT1miRNA seq1.1a, lane 4: cells transfected with BCAT1 miRNA seq2.1a, lane5: cells transfected with BCAT1 miRNA seq3.1a, lane 6: cells transfectedwith miRNA BCAT1 seq4.1a, lane 7: cells transfected with BCAT1 miRNAseq5.1a, lane 8: cells transfected with negative control miRNA. Proteinsize: 42 kD for B-Actin and 43 kD for BCAT1

FIGS. 35A and 35B: Western blot for probing the levels of BCAT1 andB-Actin in stable CHO cell pool generated from independent transfectionswith five miRNAs against BCAT1. (A) B-Actin protein levels in stablepools generated from transfections into CHO parental cell line (B) BCAT1protein levels in stable pools generated from transfections into CHOparental cell line. Gel loading details for all the gels are as follows.Lane 1: protein ladder, lane 2: cells transfected with BCAT1 miRNAseq1.1a, lane 3: cells transfected with BCAT1 miRNA seq2.1a, lane 4:cells transfected with BCAT1 miRNA seq3.1a, lane 5: cells transfectedwith miRNA BCAT1 seq4.1a, lane 6: cells transfected with BCAT1 miRNAseq5.1a, lane 7: cells transfected with negative control miRNA. Proteinsize: 42 kD for B-Actin and 43 kD for BCAT1

DETAILED DESCRIPTION

The present invention provides methods and media for cell culture. Thepresent invention provides cell culture methods where the concentrationof at least one metabolite selected from 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate, phenyllactate, phenylpyruvate, indolelactate(indole-3-lactate), indolecarboxylic acid (indole-3-carboxylate),homocysteine, 2-hydroxybutyric acid, isovalerate, 2-methyl butyrate,isobutyrate and formate, and/or at least one amino acid selected fromphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine and glycine is maintained at low levels in the cell culturemedium.

The inventors have unexpectedly discovered that, in cell culture, and inparticular in high density cell culture, such as for example fed-batchcell culture aiming at producing high amount of a recombinant protein ofinterest, the growth of cells was inhibited by the accumulation ofmetabolites such as 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate,phenyllactate, phenylpyruvate, indolelactate, indolecarboxylic acid,homocysteine, 2-hydroxybutyric acid, isovalerate, 2-methyl butyrate,isobutyrate and formate in the cell culture medium. The inhibitoryeffect of these metabolites can be limited by maintaining theirconcentration in the cell culture medium below levels where they inhibitcell growth. The inhibitory effects of these metabolites have also becan be limited by modifying one or more genes in the cell to reduce thelevel of synthesis of growth and/or productivity inhibitors by the cell,in particular where the one or more modified genes is selected fromBcat1, Bcat2, Auh, Mccc1, Mccc2, Ivd, Hpd, Hgd, Pah, PCBD1, and QDPR.

Methods Comprising Controlling the Metabolite Concentration in the CellCulture Medium at Low Levels

In a first aspect the invention provides a method of cell culturecomprising (i) providing cells in a cell culture medium to start a cellculture process, wherein the cells are modified to reduce the level ofsynthesis of growth and/or productivity inhibitors by the cell.

In some embodiments, the cells are modified to modify expression of oneor more genes, in one embodiment the genes are in the cell metabolicpathway or pathways which synthesise the growth inhibitors or metabolicintermediates thereof, or metabolites of growth inhibitors or metabolicintermediates thereof, or metabolite of the metabolic intermediate.

“Metabolic intermediates” are understood to comprise molecules which arethe precursors or metabolites required for the synthesis of a cellgrowth and/or productivity inhibitor and include reactant, product andcofactor molecules of enzymes of the cell metabolic pathway or pathways.Relevant cofactors include for example BH4 (tetrahydrobiopterin), orBH4-4a (carbinolamine), or q-BH2. Metabolic intermediates may besynthesised in the same pathway(s) as the cell metabolic pathway orpathways encoding an enzyme which catalyses the synthesis of the cellgrowth and/or productivity inhibitor or may be synthesised in abranching pathway. “Metabolites” are understood to comprise the productsof metabolic reactions catalysed by the enzymes of the cell metabolicpathway or pathways and include reactant, product and cofactor moleculesof said enzymes such as for example BH4 (tetrahydrobiopterin), or BH4-4a(carbinolamine), or q-BH2. Metabolites may arise in the same pathway(s)as the cell metabolic pathway or pathways encoding an enzyme whichcatalyses the synthesis of the cell growth and/or productivity inhibitoror intermediate thereof or may be synthesised in a branching pathway.The branching pathway may arise at a node situated above or below theone or more one or more genes in the cell metabolic pathway or pathwaysencoding an enzyme which catalyses the synthesis of the cell growthand/or productivity inhibitor or intermediate thereof.

In some embodiments, the one or more genes modified encodes an enzymethat catalyses the synthesis of 3-(4-hydroxyphenyl)lactate (HPLA),4-hydroxyphenylpyruvate, phenyllactate (PLA), phenyl pyruvate,indole-3-carboxylate (indolecarboxylic acid), indole-3-lactate(indolelactate), 2-hydroxybutyric acid, homocysteine, isovalerate,2-methyl butyrate, isobutyrate, butyrate, formate, or a metabolicintermediate thereof or metabolites of said molecules or metabolicintermediates thereof, or metabolite of the metabolic intermediate.

In some embodiments, the one or more genes modified encodes an enzymethat catalyses the synthesis of 4-hydroxyphenylpyruvate or phenyllactate(PLA), metabolite thereof or a metabolic intermediate thereof, ormetabolite of the metabolic intermediate.

In some embodiments, the one or more genes modified encodes an enzymethat catalyses the synthesis of isovalerate, 2-methylbutyrate,isobutyrate or butyrate, metabolite thereof or a metabolic intermediatethereof, or metabolite of the metabolic intermediate.

In some embodiments, the one or more genes modified is selected from;PCDB1, QDPR, Pah, Mif, Got1, Got2, Nup62-il4i1, Hpd, Hgd, Gstz1, Fah.

In some embodiments, the one or more genes modified is selected from;PCDB1, QDPR Hpd, Hgd and Pah; in one embodiment wherein the one or moregenes modified is selected from; (i) PCDB1, (ii) Pah, (iii) QDPR, (iv)PCBD1 and QDPR, (v) PCBD1 and Pah, (vi) Pah and QDPR, (vii) PCDB1 andPah, and QDPR, (viii) any of (i) to (vii) and Hpd and/or Hgd.

In some embodiments, the one or more genes modified is selected from;Bcat1, Bcat2, Bckdha/b, Dbt/Dld, Ivd, Acadm, Mccc1, Mccc2, Auh, Hmgcl,Fasn.

In some embodiments, the one or more genes modified is selected from,Bcat1, Bcat2, Auh, Mccc1, Mccc2, Ivd.

In some embodiments, the one or more genes modified is;

-   -   (i) Auh,    -   (ii) Bcat1,    -   (iii) Bcat2    -   (iv) one or more of Mccc1, Mccc2 and Ivd,    -   (v) Bcat1 and/or Bcat2 and/or Auh and one or more Mccc1, Mccc2        and Ivd,    -   (vi) Pah,    -   (vii) PCBD1,    -   (viii) PCBD1 and/or QDPR    -   (ix) one or more of Hpd and Hgd,    -   (x) Pah and/or PCBD1 and one or more of Hpd and Hgd.    -   (xi) Pah and PCBD1, and optionally QDPR    -   (xii) Pah, PCBD1, Hpd and Hgd, and optionally QDPR    -   (xiii) Bcat1 and/or Bcat2, Auh, Mccc1, Mccc2 and Ivd or    -   (ix) Bcat1 and/or Bcat2, Auh, Mccc1, Mccc2, Ivd, Pah, PCBD1,        Hpd, Hgd and optionally QDPR.

In some embodiments, the one or more genes are modified to increase ordecrease gene expression.

In some embodiments, the one or more genes are modified to increase geneexpression, in one embodiment wherein the one or more genes are Pahand/or PCDB1 and/or QDPR and/or Hpd and/or Hgd.

In some embodiments, the one or more genes are modified to decrease geneexpression, in one embodiment wherein the one or more genes are Bcat1and/or Bcat2.

In some embodiments, the one or more genes modified is selected from;(i) PCDB1, (ii) Pah, (iii) QDPR, (iv) PCBD1 and QDPR, (v) PCBD1 and Pah,(vi) Pah and QDPR, (vii) PCDB1 and Pah, and QDPR, (viii) any of (i) to(vii) and Hpd and/or Hgd.

In some embodiments, the cell comprises;

-   -   (i) an expressible nucleic acid or vector construct comprising a        PCDB1 gene,    -   (ii) an expressible nucleic acid or vector construct comprising        a Pah gene,    -   (iii) an expressible nucleic acid or vector construct comprising        a Pah gene and PCDB1 gene,    -   (iv) an expressible nucleic acid or vector construct comprising        a PCDB1 gene or Pah gene or Pah gene and PCDB1 gene, and        additionally a QDPR gene,    -   (v) an expressible nucleic acid or vector construct of (i)        to (iv) further comprising a Hpd gene and/or a Hgd gene.

In some embodiments the method further comprises;

(ii) maintaining at least one metabolite selected from3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate (indole-3-lactate), indolecarboxylic acid(indole-3-carboxylate), homocysteine, 2-hydroxybutyric acid,isovalerate, 2-methylbutyrate, isobutyrate and formate below aconcentration C1 in the cell culture medium, wherein C1 is 3 mM.

In some embodiments, C1 is 2.5 mM, 2 mM, 1.5 mM, 1 mM, 0.9 mM, 0.8 mM,0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM or 0.1 mM. In someembodiments, C1 is 1 mM. In some embodiments, C1 is 0.5 mM.

In some embodiments, step (ii) comprises the step of measuring theconcentration of said at least one metabolite.

In some embodiments, when the measured concentration is above apredefined value, the concentration of precursor of said at least onemetabolite in the cell culture medium is decreased by reducing theamount of precursor provided to the cells. Said predefined value isselected so that the decrease of concentration of said precursorprevents the concentration of metabolite from rising above C1. Thepredefined value can be equal to C1 or can be a percentage of C1. Insome embodiments the percentage is 50, 55, 60, 65, 70, 75, 80, 85, 90 or95% of C1. In some embodiments the percentage is 80% of C1.

In some embodiments, when the measured concentration of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate and/or phenyllactateis above said predefined value, the concentration of phenylalanine isdecreased in the cell culture medium.

In some embodiments, when the measured concentration of3-(4-hydroxyphenyl)lactate and/or 4-hydroxyphenylpyruvate is above saidpredefined value, the concentration of tyrosine is decreased in the cellculture medium.

In some embodiments, when the measured concentration of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate and/or phenyllactateis above said predefined value, the concentrations of tyrosine andphenylalanine are decreased in the cell culture medium.

In some embodiments, when the measured concentration of indolelactateand/or indolecarboxylic acid is above said predefined value, theconcentration of tryptophan is decreased in the cell culture medium.

In some embodiments, when the measured concentration of homocysteineand/or 2-hydroxybutyric acid is above said predefined value, theconcentration of methionine is decreased in the cell culture medium.

In some embodiments, when the measured concentration of isovalerate isabove said predefined value, the concentration of leucine is decreasedin the cell culture medium.

In some embodiments, when the measured concentration of 2-methylbutyrateis above said predefined value, the concentration of isoleucine isdecreased in the cell culture medium.

In some embodiments, when the measured concentration of isobutyrate isabove said predefined value, the concentration of valine is decreased inthe cell culture medium.

In some embodiments, when the measured concentration of formate is abovesaid predefined value, the concentration of serine, threonine and/orglycine is decreased in the cell culture medium.

In some embodiments, when the measured concentration of formate is abovesaid predefined value, the concentration of serine is decreased in thecell culture medium.

In some embodiments, when the measured concentration of formate is abovesaid predefined value, the concentration of threonine is decreased inthe cell culture medium.

In some embodiments, when the measured concentration of formate is abovesaid predefined value, the concentration of glycine is decreased in thecell culture medium.

The concentration of precursor in the cell culture medium can bedecreased by reducing the amount of precursor provided to the cells, forexample by reducing the concentration of said precursor in the feedmedium, reducing the feed rate, or reducing the number or volume offeeds. For example, the feed medium can be replaced by a feed mediumcomprising a lower concentration of precursor.

In some embodiments of the above described methods, step (ii) comprisesmaintaining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate (indole-3-lactate), indolecarboxylic acid(indole-3-carboxylate), homocysteine, 2-hydroxybutyric acid,isovalerate, 2-methylbutyrate, isobutyrate and formate below C1 in thecell culture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining 1, 2, 3, 4, 5, 6, or 7 of 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate, phenyllactate, indolelactate, indolecarboxylicacid, homocysteine and 2-hydroxybutyric acid below C1 in the cellculture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate andphenyllactate below C1 in the cell culture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining indolelactate (indole-3-lactate) and indolecarboxylic acid(indole-3-carboxylate) below C1 in the cell culture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining homocysteine and 2-hydroxybutyric acid below C1 in the cellculture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining isovalerate, 2-methylbutyrate, and isobutyrate below C1 inthe cell culture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining isobutyrate below C1 in the cell culture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining 2-methylbutyrate below C1 in the cell culture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining isovalerate below C1 in the cell culture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining formate below C1 in the cell culture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining isovalerate and 4-hydroxyphenylpyruvate below C1 in the cellculture medium.

In some embodiments of the above described methods, step (ii) comprisesmaintaining 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate,phenyllactate, indolelactate (indole-3-lactate), indolecarboxylic acid(indole-3-carboxylate), homocysteine, 2-hydroxybutyric acid isovalerate,2-methylbutyrate, isobutyrate and formate below C1 in the cell culturemedium.

In some embodiments of the above described methods, in step (ii), theconcentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.5 mM,0.4 mM, 0.3 mM, 0.2 mM, or 0.1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.3 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of 4-hydroxyphenylpyruvate is maintained below 0.1 mM,0.08 mM, 0.06 mM, 0.05 mM, 0.04 mM, 0.03 mM or 0.02 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of 4-hydroxyphenylpyruvate is maintained below 0.05 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of 4-hydroxyphenylpyruvate is maintained below 0.02 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of phenyllactate is maintained below 0.5 mM, 0.4 mM, 0.3mM, 0.2 mM, or 0.1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of phenyllactate is maintained below 0.2 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of phenyllactate is maintained below 0.1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of indolelactate (indole-3-lactate), is maintained below 3mM, 2 mM, 1 mM, 0.5 mM, 0.3 mM or 0.1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of indolelactate is maintained below 1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of indolelactate is maintained below 0.3 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of indolelactate is maintained below 0.1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of indolecarboxylic acid (indole-3-carboxylate), ismaintained below 1 mM, 0.8 mM, 0.6 mM, 0.4 mM or 0.2 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of indolecarboxylic acid is maintained below 0.5 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of indolecarboxylic acid is maintained below 0.2 mM.

In some embodiments of the above the above described methods, in step(ii), the concentration of homocysteine is maintained below 0.5 mM, 0.4mM, 0.3 mM, 0.2 mM, or 0.1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of homocysteine is maintained below 0.3 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of homocysteine is maintained below 0.1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of 2-hydroxybutyric acid is maintained below 1 mM, 0.8 mM,0.6 mM, 0.4 mM or 0.2 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of 2-hydroxybutyric acid is maintained below 0.5 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of 2-hydroxybutyric acid is maintained below 0.2 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of isovalerate and/or, 2-methylbutyrate, and/orisobutyrate is maintained below 2 mM, 1 mM, 0.8 mM, 0.6 mM, 0.4 mM or0.2 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of isovalerate and/or, 2-methylbutyrate, and/orisobutyrate is maintained below 1 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of isovalerate and/or, 2-methylbutyrate, and/orisobutyrate is maintained below 0.5 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of formate is maintained below 4 mM, 3 mM, 2 mM, 1 mM, 0.5mM or 0.2 mM.

In some embodiments of the v, in step (ii), the concentration of formateis maintained below 3 mM.

In some embodiments of the above described methods, in step (ii), theconcentration of formate is maintained below 1 mM.

Methods Comprising Controlling the Amino Acid Concentration in the CellCulture Medium at Low Levels

In some embodiments, the method of cell culture further comprises;

(iii) maintaining at least one amino acid selected from phenylalanine,tyrosine, tryptophan, methionine, leucine, isoleucine, valine, serine,threonine and glycine below a concentration C2 in the cell culturemedium, wherein C2 is 2 mM. In some embodiments, said concentration ismaintained between 0.1 mM and C2, 0.2 mM and C2, 0.3 mM and C2, 0.4 mMand C2, or 0.5 mM and C2. In some embodiments, said concentration ismaintained between 0.5 mM and C2.

In some embodiments, C2 is 2 mM, 1.5 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM,0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM, 0.05 mM. In someembodiments, C2 is 1 mM.

Methods Comprising Measuring the Amino Acid Concentration in the CellCulture Medium

In some embodiments, step (iii) comprises the step of measuring theconcentration of said at least one amino acid. The concentration ofamino acid can be measured by any method known to the skilled person,including off line and on line measurement methods.

The concentration of amino acids can be measured once or several timesduring the cell culture. In some embodiments, the amino acidconcentration is measured continuously, intermittently, every 30 min,every hour, every two hours, twice a day, daily, or every two days. In apreferred embodiment, the concentration of amino acid is measured daily.

The concentration of amino acid can be measured by any method known tothe skilled person. Preferred methods to measure the concentration ofamino acids in online or offline methods include for example LiquidChromatography such HPLC, UPLC or LCMS, NMR or GCMS.

In some embodiments, the concentration of amino acid is measured offline by taking a sample of the cell culture medium and measuring theconcentration of said at least one amino acid in said sample. In someembodiments, the concentration of amino acid is measured as disclosed inExample 4. A preferred method to measure the concentration of aminoacids in an off line method is UPLC.

In some embodiments, the concentration of amino acid is measured online.In some embodiments, the concentration of amino acid is measured on-lineusing Raman spectroscopy. In some embodiments, the concentration ofamino acid is measured on-line using Raman spectroscopy as disclosed inExample 7. In some embodiments, the concentration of amino acid ismeasured online using HPLC or UPLC based technology with an auto-samplerthat draws sample from reactor and transfers to the equipment in aprogrammed manner.

In some embodiments, when the measured concentration is above apredefined value, the concentration of said at least one amino acid inthe cell culture medium is decreased. The predefined value can be equalC2 or can be a percentage of C2. In some embodiments the percentage is50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% of C2. In some embodiments thepercentage is 80% of C2.

The concentration of amino acid in the cell culture medium can bedecreased by reducing the amount of amino acid provided to the cells,for example by reducing the concentration of said amino acid in the feedmedium, reducing the feed rate, or reducing the number or volume offeeds. For example, the feed medium can be replaced by a feed mediumcomprising a lower concentration of amino acid.

Concentration of Phenylalanine, Tyrosine, Tryptophan Methionine,Leucine, Isoleucine, Valine, Serine, Threonine and Glycine in the CellCulture Medium

In some embodiments, in step (iii), the concentration of phenylalanineis maintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of phenylalanine is maintained between 0.1and 2 mM in the cell culture medium. In a preferred embodiment, theconcentration of phenylalanine is maintained between 0.1 and 1 mM in thecell culture medium. In a preferred embodiment, the concentration ofphenylalanine is maintained between 0.2 and 1 mM in the cell culturemedium. In a preferred embodiment, the concentration of phenylalanine ismaintained between 0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of tyrosine ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of tyrosine is maintained between 0.1 and2 mM in the cell culture medium. In a preferred embodiment, theconcentration of tyrosine is maintained between 0.1 and 1 mM in the cellculture medium. In a preferred embodiment, the concentration of tyrosineis maintained between 0.2 and 1 mM in the cell culture medium. In apreferred embodiment, the concentration of tyrosine is maintainedbetween 0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of tryptophan ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of tryptophan is maintained between 0.1and 2 mM in the cell culture medium. In a preferred embodiment, theconcentration of tryptophan is maintained between 0.1 and 1 mM in thecell culture medium. In a preferred embodiment, the concentration oftryptophan is maintained between 0.2 and 1 mM in the cell culturemedium. In a preferred embodiment, the concentration of tryptophan ismaintained between 0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of methionine ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of methionine is maintained between 0.1and 2 mM in the cell culture medium. In a preferred embodiment, theconcentration of methionine is maintained between 0.1 and 1 mM in thecell culture medium. In a preferred embodiment, the concentration ofmethionine is maintained between 0.2 and 1 mM in the cell culturemedium. In a preferred embodiment, the concentration of methionine ismaintained between 0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of leucine ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of leucine is maintained between 0.1 and 2mM in the cell culture medium. In a preferred embodiment, theconcentration of leucine is maintained between 0.1 and 1 mM in the cellculture medium. In a preferred embodiment, the concentration of leucineis maintained between 0.2 and 1 mM in the cell culture medium. In apreferred embodiment, the concentration of leucine is maintained between0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of isoleucine ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of isoleucine is maintained between 0.1and 2 mM in the cell culture medium. In a preferred embodiment, theconcentration of isoleucine is maintained between 0.1 and 1 mM in thecell culture medium. In a preferred embodiment, the concentration ofisoleucine is maintained between 0.2 and 1 mM in the cell culturemedium. In a preferred embodiment, the concentration of isoleucine ismaintained between 0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of valine ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of valine is maintained between 0.1 and 2mM in the cell culture medium. In a preferred embodiment, theconcentration of valine is maintained between 0.1 and 1 mM in the cellculture medium. In a preferred embodiment, the concentration of valineis maintained between 0.2 and 1 mM in the cell culture medium. In apreferred embodiment, the concentration of valine is maintained between0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of serine ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of serine is maintained between 0.1 and 2mM in the cell culture medium. In a preferred embodiment, theconcentration of serine is maintained between 0.1 and 1 mM in the cellculture medium. In a preferred embodiment, the concentration of serineis maintained between 0.2 and 1 mM in the cell culture medium. In apreferred embodiment, the concentration of serine is maintained between0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of threonine ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of threonine is maintained between 0.1 and2 mM in the cell culture medium. In a preferred embodiment, theconcentration of threonine is maintained between 0.1 and 1 mM in thecell culture medium. In a preferred embodiment, the concentration ofthreonine is maintained between 0.2 and 1 mM in the cell culture medium.In a preferred embodiment, the concentration of threonine is maintainedbetween 0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of glycine ismaintained below 2 mM in the cell culture medium. In a preferredembodiment, the concentration of glycine is maintained between 0.1 and 2mM in the cell culture medium. In a preferred embodiment, theconcentration of glycine is maintained between 0.1 and 1 mM in the cellculture medium. In a preferred embodiment, the concentration of glycineis maintained between 0.2 and 1 mM in the cell culture medium. In apreferred embodiment, the concentration of glycine is maintained between0.5 and 1 mM in the cell culture medium.

In some embodiments, in step (iii), the concentration of tyrosine,phenylalanine and leucine is maintained below 2 mM, preferably between0.1 and 2 mM, between 0.1 and 1 mM, between 0.2 and 1 mM or between 0.5and 1 mM in the cell culture medium.

In some embodiments the cell culture medium comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, of glycine, proline, serine, threonine, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 1 mM, 1.5 mM, 2 mM, 3 mM or 5 mM.

In some embodiments, the cell culture medium comprises one of glycine,proline, serine, threonine, lysine, arginine, histidine, aspartate,glutamate and asparagine at a concentration above 2 mM.

In some embodiments, the cell culture medium comprises one of glycine,proline, serine, threonine, lysine, arginine, histidine, aspartate,glutamate and asparagine at a concentration above 5 mM.

In some embodiments, the cell culture medium comprises 1, 2, 3, 4, 5, 6,or 7, of, proline, lysine, arginine, histidine, aspartate, glutamate andasparagine at a concentration above 1 mM, 1.5 mM, 2 mM, 3 mM or 5 mM.

In some embodiments, the cell culture medium comprises one of, proline,lysine, arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM.

In some embodiments, the cell culture medium comprises one of, proline,lysine, arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 5 mM.

In some embodiments, the cell culture medium comprises no tyrosine. Insome embodiments, the cell culture is a HiPDOG culture wherein themedium comprises no tyrosine. In some embodiments, the cell culturemedium is HiPDOG medium, medium used in the HipDOG process, butcomprising no tyrosine. In some embodiments the cell culture mediumcomprises no tyrosine wherein a HiPDOG culture or process is used whichcomprises no tyrosine.

Concentration of Lactate and Ammonia

In some embodiments of, other metabolites inhibiting growth of cells,such as lactate and ammonia are also maintained at low levels in thecell culture medium.

Methods to keep lactate and ammonia at low levels are known to theskilled person.

For example, lactate can be kept at low levels in cell culture by usingmethods disclosed in WO2004104186, Gagnon et al, Biotechnology andBioengineering, Vol. 108, No. 6, June, 2011 (Gagnon et Al) orWO2004048556.

Various other strategies can be employed to restrict lactate productionand/or induce lactate consumption. These include culturing cells underslightly reduced pH (6.7-7.0), culturing cells at low glucoseconcentrations by using alternative carbon sources including but notlimited to fructose (Wlaschin & Hu, 2007) and galactose (Altamirano etal, 2006), using a cell line that has reduced protein levels ofglycolytic enzymes including but not limited to hexose transporter orlactate dehydrogenase (Kim & Lee, 2007a), employing a cell line withsuppressed cellular protein levels of both lactate dehydrogenase andpyruvate dehydrogenase kinase (Zhou et al, 2011), or cell line withover-expression of pyruvate carboxylase enzyme (Kim & Lee, 2007b), orwith the use of inhibitors (small molecule or protein based) forsignaling pathways (such as AKT (Mulukutla et al, 2012), mTOR (Duvel etal, 2010; Lee & Lee, 2012), HIF1a) that regulate the activity of energymetabolism pathways (glycolysis, TCA cycle, and redox pathway).

In some embodiments lactate is maintained at low levels by using thehigh-end pH-controlled delivery of glucose (HIPDOG process/HIPDOGculture) disclosed in Gagnon et al.

In some embodiments of lactate is maintained at low levels in the cellculture medium. In some embodiments, concentration of lactate in thecell culture medium is maintained below 90 mM. In a preferredembodiment, the concentration of lactate in the cell culture medium ismaintained below 70 mM. In some embodiments, the concentration oflactate in the cell culture medium is maintained below 50 mM. In someembodiments, the concentration of lactate in the cell culture medium ismaintained below 40 mM. In some embodiments, lactate is maintained atlow levels by controlling the amount of glucose provided to the cellculture. In some embodiments, lactate is maintained at low levels byusing the HIPDOG process/culture. In some embodiments, a pH sensor isused to monitor pH of the cell culture, and, in response to a rise abovea predetermined pH value, glucose is fed to the cell culture. In someembodiments, the predetermined pH value is approximately 7.

Ammonia can be kept at low levels in cell culture by any method known tothe skilled person such as for example the methods disclosed in Butleret Al, Cytotechnology 15: 87-94, 1994 or Hong et Al, Appl MicrobiolBiotechnol (2010) 88:869-876. Alternatively, ammonia can be kept at lowlevels by using a glutamine synthetase (GS) expression system. Suchsystems are commercially available (Lonza) and can be used to generaterecombinant cell lines. Cell lines using GS expression systemdemonstrate the gain of function to synthesize glutamine in vivo,thereby completely relieving cellular dependence on the externallysupplied glutamine. Since the major fraction of ammonia produced inculture is from the catabolysis of externally supplied glutamine, such again of metabolic function reduces the levels of ammonia produced inculture.

In some embodiments of any of the ammonia is maintained at low levels inthe cell culture medium. In a preferred embodiment, concentration ofammonia in the cell culture medium is maintained below 20 mM. In apreferred embodiment, the concentration of ammonia in the cell culturemedium is maintained below 10 mM. In a preferred embodiment, theconcentration of ammonia in the cell culture medium is maintained below8 mM.

Cell Culture Methods

The terms “culture” and “cell culture” as used herein refer to a cellpopulation that is suspended in a medium under conditions suitable tosurvival and/or growth of the cell population. As will be clear to thoseof ordinary skill in the art, in some embodiments, these terms as usedherein refer to the combination comprising the cell population and themedium in which the population is suspended. In some embodiments, thecells of the cell culture comprise mammalian cells.

The present invention may be used with any cell culture method that isamenable to the desired process (e.g., production of a recombinantprotein (e.g., antibody)). As a non-limiting example, cells may be grownin batch or fed-batch cultures, where the culture is terminated aftersufficient expression of the recombinant protein (e.g., antibody), afterwhich the expressed protein (e.g., antibody) is harvested.Alternatively, as another non-limiting example, cells may be grown inbatch-refeed, where the culture is not terminated and new nutrients andother components are periodically or continuously added to the culture,during which the expressed recombinant protein (e.g., antibody) isharvested periodically or continuously. Other suitable methods (e.g.,spin-tube cultures) are known in the art and can be used to practice thepresent invention.

In some embodiments, a cell culture suitable for the present inventionis a fed-batch culture. The term “fed-batch culture” as used hereinrefers to a method of culturing cells in which additional components areprovided to the culture at a time or times subsequent to the beginningof the culture process. Such provided components typically comprisenutritional components for the cells which have been depleted during theculturing process. A fed-batch culture is typically stopped at somepoint and the cells and/or components in the medium are harvested andoptionally purified. In some embodiments, the fed-batch culturecomprises a base medium supplemented with feed media.

Cells may be grown in any convenient volume chosen by the practitioner.For example, cells may be grown in small scale reaction vessels rangingin volume from a few milliliters to several liters. Alternatively, cellsmay be grown in large scale commercial Bioreactors ranging in volumefrom approximately at least 1 liter to 10, 50, 100, 250, 500, 1000,2500, 5000, 8000, 10,000, 12,000, 15000, 20000 or 25000 liters or more,or any volume in between.

The temperature of a cell culture will be selected based primarily onthe range of temperatures at which the cell culture remains viable andthe range in which a high level of desired product (e.g., a recombinantprotein) is produced. In general, most mammalian cells grow well and canproduce desired products (e.g., recombinant proteins) within a range ofabout 25° C. to 42° C., although methods taught by the presentdisclosure are not limited to these temperatures. Certain mammaliancells grow well and can produce desired products (e.g., recombinantproteins or antibodies) within the range of about 35° C. to 40° C. Incertain embodiments, a cell culture is grown at a temperature of 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44 or 45° C. at one or more times during the cellculture process. Those of ordinary skill in the art will be able toselect appropriate temperature or temperatures in which to grow cells,depending on the particular needs of the cells and the particularproduction requirements of the practitioner. The cells may be grown forany amount of time, depending on the needs of the practitioner and therequirement of the cells themselves. In some embodiment, the cells aregrown at 37° C. In some embodiments, the cells are grown at 36.5° C.

In some embodiments, the cells may be grown during the initial growthphase (or growth phase) for a greater or lesser amount of time,depending on the needs of the practitioner and the requirement of thecells themselves. In some embodiments, the cells are grown for a periodof time sufficient to achieve a predefined cell density. In someembodiments, the cells are grown for a period of time sufficient toachieve a cell density that is a given percentage of the maximal celldensity that the cells would eventually reach if allowed to growundisturbed. For example, the cells may be grown for a period of timesufficient to achieve a desired viable cell density of 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percentof maximal cell density. In some embodiments, the cells are grown untilthe cell density does not increase by more than 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% per day of culture. In someembodiments, the cells are grown until the cell density does notincrease by more than 5% per day of culture.

In some embodiment the cells are allowed to grow for a defined period oftime. For example, depending on the starting concentration of the cellculture, the temperature at which the cells are grown, and the intrinsicgrowth rate of the cells, the cells may be grown for 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days,preferably for 4 to 10 days. In some cases, the cells may be allowed togrow for a month or more. The practitioner of the present invention willbe able to choose the duration of the initial growth phase depending onprotein production requirements and the needs of the cells themselves.

The cell culture may be agitated or shaken during the initial culturephase in order to increase oxygenation and dispersion of nutrients tothe cells. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor during theinitial growth phase, including but not limited to pH, temperature,oxygenation, etc.

At the end of the initial growth phase, at least one of the cultureconditions may be shifted so that a second set of culture conditions isapplied and a metabolic shift occurs in the culture. A metabolic shiftcan be accomplished by, e.g., a change in the temperature, pH,osmolality or chemical inductant level of the cell culture. In onenon-limiting embodiment, the culture conditions are shifted by shiftingthe temperature of the culture. However, as is known in the art,shifting temperature is not the only mechanism through which anappropriate metabolic shift can be achieved. For example, such ametabolic shift can also be achieved by shifting other cultureconditions including, but not limited to, pH, osmolality, and sodiumbutyrate levels. The timing of the culture shift will be determined bythe practitioner of the present invention, based on protein productionrequirements or the needs of the cells themselves.

When shifting the temperature of the culture, the temperature shift maybe gradual. For example, it may take several hours or days to completethe temperature change. Alternatively, the temperature shift may beabrupt. For example, the temperature change may be complete in less thanseveral hours. Given the appropriate production and control equipment,such as is standard in the commercial large-scale production ofpolypeptides or proteins, the temperature change may even be completewithin less than an hour.

In some embodiments, once the conditions of the cell culture have beenshifted as discussed above, the cell culture is maintained for asubsequent production phase under a second set of culture conditionsconducive to the survival and viability of the cell culture andappropriate for expression of the desired polypeptide or protein atcommercially adequate levels.

As discussed above, the culture may be shifted by shifting one or moreof a number of culture conditions including, but not limited to,temperature, pH, osmolality, and sodium butyrate levels. In someembodiments, the temperature of the culture is shifted. According tothis embodiment, during the subsequent production phase, the culture ismaintained at a temperature or temperature range that is lower than thetemperature or temperature range of the initial growth phase. Asdiscussed above, multiple discrete temperature shifts may be employed toincrease cell density or viability or to increase expression of therecombinant protein.

In some embodiments, the cells may be maintained in the subsequentproduction phase until a desired cell density or production titer isreached. In another embodiment of the present invention, the cells areallowed to grow for a defined period of time during the subsequentproduction phase. For example, depending on the concentration of thecell culture at the start of the subsequent growth phase, thetemperature at which the cells are grown, and the intrinsic growth rateof the cells, the cells may be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, thecells may be allowed to grow for a month or more. The practitioner ofthe present invention will be able to choose the duration of thesubsequent production phase depending on polypeptide or proteinproduction requirements and the needs of the cells themselves.

The cell culture may be agitated or shaken during the subsequentproduction phase in order to increase oxygenation and dispersion ofnutrients to the cells. In accordance with the present invention, one ofordinary skill in the art will understand that it can be beneficial tocontrol or regulate certain internal conditions of the bioreactor duringthe subsequent growth phase, including but not limited to pH,temperature, oxygenation, etc.

In some embodiments, the cells express a recombinant protein and thecell culture method of the invention comprises a growth phase and aproduction phase.

In some embodiments of the method of cell culture, step (ii) and/or(iii) is applied during the totality of the cell culture method. In someembodiments step (ii) and/or (iii) is applied during a part of the cellculture method. In some embodiments, step (ii) and/or (iii) is applieduntil a predetermined viable cell density is obtained. In someembodiments, the cell culture method of the invention comprises a growthphase and a production phase and step (ii) and/or (iii) is appliedduring the growth phase. In some embodiments, the cell culture method ofthe invention comprises a growth phase and a production phase and step(ii) and/or (iii) is applied during a part of the growth phase. In someembodiments, the cell culture method of the invention comprises a growthphase and a production phase and step (ii) and/or (iii) is appliedduring the growth phase and the production phase. In step (ii) and/or(iii), the term “maintaining” can refer to maintaining the concentrationof amino acid or metabolite below C1 or C2 for the entire cultureprocess (until harvesting) or for a part of the culture process such asfor example the growth phase, a part of the growth phase or until apredetermined cell density is obtained.

The present invention also provides a method of producing cells withimproved cell growth and/or productivity in cell culture comprising thesteps of:

(i) identification of a cell metabolite or metabolites which are cellgrowth and/or productivity inhibitors,

(ii) identification of the cell metabolic pathway or pathways resultingin the synthesis of the cell growth and/or productivity inhibitors,

(iii) identification of one or more genes in the cell metabolic pathwayor pathways encoding an enzyme which catalyses the synthesis of the cellgrowth and/or productivity inhibitors or a metabolic intermediatesthereof, or one or more genes encoding an enzyme in metabolic pathwaysbranching from or directly branching therefrom, and/or(iv) Identification of one or more genes in the cell metabolic pathwayor pathways encoding an enzyme which catalyses the synthesis of the cellgrowth and/or productivity inhibitors, or metabolites of the cell growthand/or productivity inhibitors or metabolic intermediate(s) of the cellgrowth and/or productivity inhibitors, or metabolite of the metabolicintermediate(s), or cofactors in the cell metabolic pathway or pathwaysor BH4 (tetrahydrobiopterin), or BH4-4a (carbinolamine), or q-BH2, and(v) modifying the expression of the one or more genes to reduce thelevel of synthesis of the cell growth and/or productivity inhibitors.

In some embodiments the identification of cell metabolites which arecell growth and/or productivity inhibitors comprises;

(i) measuring the concentration/level of measurable cell metabolitesproduced during cell culture up to maximum viable cell density,

(ii) identifying the cell metabolites which are highly expressedmetabolites or metabolites that accumulate to high levels/concentrationsand/or which demonstrate an increased rate of metabolite production oran increased level/concentration of metabolite production during thecell culture relative to other metabolites.

Identification of cell metabolites which are highly expressedmetabolites or are metabolites that accumulate to highlevels/concentrations can be performed using methods for identifyingand/or measuring the metabolite concentration in the cell culture mediumor in the cells, for example in the cell pellet for example using one ormore of or a combination of NMR, LC/MS and GC/MS techniques. Formetabolomic analysis, spent media samples and the cell pellet samplescan be collected and analyzed for example from single or from duplicatereactors runs, performed for each condition taken at time pointsthroughout culture, for example up to and/or including maximum viablecell density, for example at time points of days selected from 0, 1, 2,3, 4, 5, 7, 8, 9, 11, and 12. The relative levels (fold changes) of allmeasurable metabolites are measured and calculated. The relative levelsare determined in both the spent media and/or cell pellet samples, andare calculated based on fold changes compared to the level of themetabolite when first detected until the desired time endpoint, usuallymaximum viable cell density. Metabolites accumulating to high levels arejudged from a fold change cut-off of any one of greater than or equal to10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 90 or 100 fold betweenthe first time point the metabolite was measured and the desired timeendpoint, for example maximum viable cell density. In some embodimentsit is greater than or equal to 20 fold.

In some embodiments the increase in rate of metabolite production or theincreased level/concentration of metabolite production is greater thanor equal 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold or more increased duringcell culture up to maximum viable cell density. In some embodiments itis greater than or equal to 15, 20, 25, 30, 35, 40, 45 or 50 fold. Insome embodiments it is greater than or equal to 40 fold. In someembodiments it is greater than or equal to 50 fold.

In some embodiments the identification of cell metabolites which arecell growth (productivity) inhibitors further comprises

(i) quantifying the highly expressed metabolite production/concentrationat maximum viable cell density,

(ii) culturing cells with and without the presence of the quantity ofhighly expressed metabolite in (i),

(iii) comparing maximum viable cell density or productivity during cellculture and/or maximum viable cell density and or productivity for eachcell population in (ii) and determining the effect of the highlyexpressed metabolite on cultured cell growth and/or productivity.

Quantifying the highly expressed metabolite production/concentration atmaximum viable cell density can be performed using methods foridentifying and/or measuring the metabolite concentration in the cellculture medium or in the cells. In some embodiments the measurement ofand/or the quantification of the metabolites, including highly expressedmetabolites, is performed using liquid chromatography with massspectrometry (LC/MS), gas chromatography with mass spectrometry (GC/MS),or nuclear magnetic resonance (NMR). In some embodiments the cellmetabolites and/or highly expressed metabolites are identified fromsamples of the culture medium or from the culture cells during cellculture.

The measurement of and/or the quantification of metabolites or highlyexpressed metabolites can be carried out by using obtained purifiedcompound forms of the metabolite or highly expressed metabolite. Thesecompounds can be used to prepare calibration curves for the compounds atknown concentrations in order to precisely correlate the measurementsobtained from the methods used with the concentrations of metabolite. Inthis way a precise measurement of the concentration of the metabolite isdeterminable at any given time point in the cell culture, for example atmaximum viable cell density.

The effect of quantified concentrations of highly expressed metabolitecan be determined on the cell growth and/or productivity of cells incell culture by introduction of the determined concentration into aculture of cells and comparing the effect on the cell growth and/orproductivity with an otherwise identical culture lacking the introducedmetabolite. For example cells, which can be cells producing arecombinant protein, can be inoculated into a culture medium spiked-inwith different selected concentrations of a highly expressed metabolite,either alone or in combination with other different highly expressedmetabolites at selected concentrations, these concentrations can be forexample the determined concentration at maximum viable cell density orserial dilutions thereof. The pH of the culture medium may also beadjusted to 7 before inoculating the cells. The comparison can determinethe existence of a negative effect of a metabolite on cell growth and/orproductivity, and/or the existence of synergistic effect betweenmetabolites on the same measures and thereby such highly expressedmetabolites can be determined to be inhibitors of cell growth and/orproductivity.

In some embodiments of the method of producing cells, the cell metabolicpathway or pathways is determined from the nutrient source or componentof the culture medium of the highly expressed metabolite or cellmetabolite or metabolites which are cell growth and/or productivityinhibitors, these fall broadly into amino acids, vitamins, inorganicsalts, trace elements, vitamins, energy sources, lipids, andnucleotides, further disclosure is provided herein. The skilled personpossessing the knowledge of the identity of a highly expressedmetabolite which is an inhibitory metabolite and knowledge of thenutrient sources for the metabolite can apply biochemical knowledge ofmetabolic pathways common to the cell to deduce the relevant cellmetabolic pathway or pathways which contribute to the synthesis of thegrowth and/or productivity inhibitors and which can include pathwaysleading to intermediates of the metabolite as well as pathways branchingtherefrom.

In some embodiments the identification of one or more genes in the cellmetabolic pathway or pathways encoding an enzyme which catalyses thesynthesis of the cell growth and/or productivity inhibitors or metabolicintermediates or one or more genes encoding an enzyme in metabolicpathways branching therefrom further comprises;

(i) determining the relative gene expression levels in the cellmetabolic pathway or pathways, and identifying genes which are expressedat an increased or decreased level, and/or

(ii) identifying genes in the cell metabolic pathway or pathways whichcomprise a mutation.

In some embodiments the identification of one or more genes in the cellmetabolic pathway or pathways encoding an enzyme which catalyses thesynthesis of the cell growth and/or productivity inhibitors or metabolicintermediates thereof or one or more genes encoding an enzyme in ametabolic pathway branching therefrom, or identification of one or moregenes in the cell metabolic pathway or pathways encoding an enzyme whichcatalyses the synthesis of the cell growth and/or productivityinhibitors, or metabolites of the cell growth and/or productivityinhibitors or metabolic intermediate(s) of the cell growth and/orproductivity inhibitors, or metabolite of the metabolic intermediate(s),or cofactors in the cell metabolic pathway or pathways for example BH4(tetrahydrobiopterin), or BH4-4a (carbinolamine), or q-BH2, and furthercomprises modifying the one or more genes. In some embodiments theidentification of the one or more genes comprises gene expressionanalysis, in some embodiments it comprises gene expression analysis ofone or more genes in the identified pathway or pathways, in someembodiments it comprises gene expression analysis of all the genes inthe identified pathway or pathways. In some embodiments it comprisesgene expression analysis by measuring transcript abundance, in someembodiments it comprises real time quantitative PCR assay or RT-qPCRassay. RT qPCR can measure transcript abundance by amplifying a targetcDNA sequence using PCR in combination with a detection reagent, forexample SYBR Green. Relative gene expression levels can be determined bymeasuring the number of PCR cycles required for the signal fromdetection reagent to surpass the background and increaselogarithmically. This cycle number is commonly referred to as the C_(T)(Threshold Cycle). Low abundance transcripts have a high C_(T) values incomparison to known control standards of transcript abundance, such asbeta-actin, and vice-versa.

In some embodiments the relative gene expression level is determined byreal time quantitative PCR, RT-qPCR, In some embodiments theidentification of gene mutation is determined by mRNA sequencing.

In some embodiments the identified gene is expressed at an increased ordecreased level higher or lower than the expression levels of a controlgene, in some embodiments the level is greater than or equal to any oneof 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75, 80, 85 90or 95 percent higher or lower than the expression levels of a controlgene, for example as judged by Ct value or dCt value. In someembodiments the identified gene is expressed 10% higher or lower thanthe expression levels of a control gene, in some embodiments 15% higheror lower, in some embodiments 20% higher or lower than the expressionlevels of a control gene. In some embodiments the control gene has a Ctvalue of equal to or between 14 and 17 and or has a Ct value of equal toor between 15 and 16 as measured by RT-qPCR, in some embodiments thecontrol gene is beta actin. In some embodiments the identified gene hasa Ct value of greater than or equal to any one of 23, 24, 25, 26, 27,28, 29, 30 or greater than 30 as measured by RT-qPCR, in someembodiments the identified gene has a Ct value of less than or equal toany one of 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less than 14as measured by RT-qPCR. In some embodiments the identified gene has adCt value of less than or equal to −1 less than or equal to 0 or lessthan or equal to 1 or more than or equal to any one of 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or 11 or more than or equal to 11 as measured by RT-qPCR.

In some embodiments the identification of one or more genes in the cellmetabolic pathway or pathways encoding an enzyme which catalyses thesynthesis of the cell growth and/or productivity inhibitors or metabolicintermediates thereof or one or more genes encoding an enzyme in ametabolic pathway branching therefrom, additionally or alternativelycomprises gene mutation analysis of one or more genes in the identifiedpathway or pathways. In some embodiments it comprises gene mutationanalysis of all the genes in the identified pathway or pathways.

In some embodiments where the one or more genes in the cell metabolicpathway or encoding an enzyme which catalyses the synthesis of the cellgrowth and/or productivity inhibitors or metabolic intermediates thereofor one or more genes encoding an enzyme in a metabolic pathway branchingtherefrom is expressed at an increased or decreased level higher orlower than the expression levels of a control gene and/or is mutated,the expression of the one or more genes is/may be modified.

In some embodiments the expression of the one or more one or more genesin the cell metabolic pathway or pathways encoding an enzyme whichcatalyses the synthesis of the cell growth and/or productivity inhibitoror metabolic intermediates thereof is modified.

In some embodiments one or more genes encoding an enzyme in a metabolicpathway branching from the cell metabolic pathway or pathways comprisingthe one or more one or more genes encoding an enzyme which catalyses thesynthesis of the cell growth and/or productivity inhibitor or metabolicintermediates thereof is modified.

In some embodiments the branch arises in a node situated above the oneor more one or more genes in the cell metabolic pathway or pathwaysencoding an enzyme which catalyses the synthesis of the cell growthand/or productivity inhibitor or metabolic intermediate thereof. In someembodiments the branch arises in a node situated below.

In some embodiments the branch arises in a node situated above the oneor more one or more gene in the cell metabolic pathway or pathwaysencoding an enzyme which catalyses the synthesis of the cell growthand/or productivity inhibitor, in some embodiments the branch arises ina node situated below.

In some embodiments one or more genes in the cell metabolic pathway orpathways encoding an enzyme which catalyses the synthesis of the cellgrowth and/or productivity inhibitors, or metabolites of the cell growthand/or productivity inhibitors or metabolic intermediate(s) of the cellgrowth and/or productivity inhibitors, or metabolite of the metabolicintermediate(s), or cofactors in the cell metabolic pathway or pathwaysfor example BH4 (tetrahydrobiopterin), or BH4-4a (carbinolamine), orq-BH2, is modified

As illustrated in the examples the method of producing cells withimproved cell growth and/or productivity in cell culture involvesidentifying the metabolite inhibitors produced by the cell. The methodfurther involves identifying metabolic pathways leading to thesynthesis, the point of inhibitor synthesis or leading away from thispoint such that metabolism is channelled to and/or from the point ofinhibitor synthesis (see FIG. 24 ). Likewise pathways branching fromthese metabolic pathways are identified (see FIG. 25 ). Such branchesmay arise at nodes in the pathway situated above, below or at the pointof inhibitor synthesis and likewise serve to channel metabolism towardsthe inhibitor and/or away from the inhibitor point of synthesis, orserve to channel metabolism towards or away from the point of synthesisof intermediates of the inhibitor synthesis. Furthermore the method asillustrated in the examples involves the identification of genesencoding enzymes in these above mentioned pathways, which includes thebranches or branching pathways. These genes may encode enzymes whichsynthesise the inhibitor; they may encode enzymes which synthesiseintermediates or upstream intermediates for the synthesis of theinhibitor; they may encode enzymes for which the inhibitor is anintermediate or an upstream intermediate for the enzyme action; or theymay encode enzymes for which the inhibitor is direct intermediate orsubstrate for the enzyme action (for example this is illustrated in FIG.24 by genes Hpd and Hgd) or may encode enzymes which generate cofactorsin the cell metabolic pathway or pathways for example BH4(tetrahydrobiopterin), or BH4-4a (carbinolamine), or q-BH2.

As illustrated in the examples the method further involves modifying theexpression of the one or more above mentioned genes to reduce the levelof synthesis of the cell growth and/or productivity inhibitors. Thisobjective can be achieved in a number of ways as the examplesillustrate. Genes encoding enzymes which synthesise the inhibitor orwhich synthesis intermediates in the pathway leading to the inhibitor orpathways branching therefrom, may be modified to reduce gene expression,hence reducing the metabolic channelling towards production of theinhibitor, particularly in the case when the one or more gene or genesis highly expressed. Genes encoding enzymes for which the inhibitor is asubstrate or an intermediate or an upstream intermediate in the pathwayleading from the inhibitor or pathways branching therefrom, may bemodified to increase gene expression, hence increasing the metabolicchannelling away from production of the inhibitor, particularly in thecase when the one or gene or genes is under expressed and/or is mutatedand/or suffers loss of function or enzyme activity. Likewise genesencoding enzymes for which an intermediate or upstream intermediate ofthe inhibitor synthesis is also an intermediate, i.e. genes in a pathwaybranching from a node located at an intermediate synthesis pointupstream of the inhibitor synthesis point, may be modified to increasegene expression, hence increasing the metabolic channelling away fromthe pathway leading to the production of the inhibitor, particularly inthe case when the one or gene or genes is under expressed and/or ismutated and/or suffers loss of function or enzyme activity.

In cases where the one or more genes identified encode an enzyme eitherdirectly synthesising the inhibitor or an intermediate to the inhibitorsynthesis or metabolites of either inhibitor or intermediate it may notbe desirable to modify such genes if they are involved in otherimportant metabolic processes, This is illustrated in Example 4 (FIG. 24) with reference to genes Got1, Got2, Nup62-il4i1 and Mif, which may beexpressed at normal or high levels. In this situation one or more genesin a branching pathway may be modified, for example in a pathwaybranching from a node located at an intermediate synthesis pointupstream of the inhibitor synthesis point, to increase the metabolicchannelling away from the pathway leading to the production of theinhibitor. This can be achieved by increasing the expression of genes inthe branch and particularly in the case when the one or more genes inthe branch is under expressed and/or is mutated and/or suffers loss offunction or enzyme activity. This illustrated in Example 4 (FIG. 24 )for gene Pah. As an alternative genes encoding enzymes for which theinhibitor is a substrate or an upstream intermediate may be modified toincrease gene expression, hence increasing the metabolic channellingaway production of the inhibitor, particularly in the case when the oneor gene or genes is under expressed and/or is mutated and/or suffersloss of function or enzyme activity. This illustrated in Example 4 (FIG.24 ) for genes Hpd and/or Hgd. Hence the metabolic targets for thephenylalanine/tyrosine pathway (FIG. 24 ) are one or more of Pah, Hpdand/or Hgd genes and/or PCDB1 and/or QDPR (FIGS. 26 and 27 ). These aretargets for increasing expression, which can be achieved by mutation ofthe gene to correct loss of activity or increase activity and/or byproviding an additional wild type copy or copies of the one or moregenes in an expressible vector which can be introduced into the cell.

In cases where the one or more genes identified encode an enzyme eitherdirectly synthesising the inhibitor or an intermediate to the inhibitorsynthesis or metabolites of either inhibitor or intermediate it maydesirable to modify such genes to prevent metabolic channelling towardsproduction of the inhibitor synthesis. As illustrated in Example 24 thiscan be achieved by modifying the one or more genes directly upstreamfrom production of the inhibitor as shown for genes Bcat1 and/or Bcat2.Such modification can be by gene knockdown or gene knockout.

In one embodiment gene knockdown reduces gene expression or activity oractivity of the encoded molecule to less than or equal to any of, 90,80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 2.5 percent level ofthe expression or activity compared to unmodified cells.

In one embodiment Bcat1 and/or Bcat2 knockdown is to less than or equalto any of, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 2.5percent level of Bcat1 and/or Bcat2 expression or activity compared tounmodified cells.

Knockdown can be achieved by any one or more of gene deletion,disruption, substitution, point mutation, multiple point mutation,insertion mutation or frameshift mutation applied to the identified geneto be expressed at a decreased level or by repression of gene expressionby use of CRISPR/CAS9 or CRISPR interference or interfering RNA,interfering mRNA or interfering aptamer, or siRNA or siRNA interferenceor a zinc finger transcription factor or a zinc finger nuclease or atranscription activator-like effector nucleases (TALEN) or by use of aninhibitor such as a an inhibitor molecule or small molecule inhibitor,for example an activity inhibitor of protein or enzyme activity.

As also illustrated in the examples increasing the metabolic channellingaway from production of the inhibitor synthesis can be achieved bymodifying one or more genes encoding enzymes which themselves share anintermediate of inhibitor production as their own intermediate. Asillustrated in Example 5 this can be achieved by modifying the one ormore genes directly downstream from, or downstream from a node whichbranches out towards, the inhibitor production (FIG. 25 ) to increaseexpression. This is particularly where the one or more genes is underexpressed or is mutated and suffers loss of function or enzyme activity.This is illustrated for genes Ivd, Mccc1 and/or Mccc2 where the enzymeshave suffered activity altering mutation and gene Auh which is underexpressed. Hence the metabolic targets for the leucine pathway are oneor more of Ivd, Mccc1, Mccc2 and Auh genes. These are targets forincreasing expression which can be achieved by mutation of the gene tocorrect loss of activity or increase activity and/or by providing a wildtype copy of the one or more genes in an expressible vector which can beintroduced into the cell.

According to some embodiments the modification suppresses, reduces,prevents the biosynthesis of the growth and/or productivity inhibitorand/or an intermediate thereof, in some embodiments the modificationsuppresses, reduces, prevents the biosynthesis of the growth and/orproductivity inhibitor. According to some embodiments the modificationproduces cells with improved cell growth and/or productivity in cellculture.

In some embodiments modifying the expression of the one or more genescomprises;

(a) any one or more of gene deletion, disruption, substitution, pointmutation, multiple point mutation, insertion mutation or frameshiftmutation applied to the identified gene which is expressed at anincreased or decreased level or identified gene which comprises amutation, or(b) introduction of one or more nucleic acids comprising the gene intothe cell, optionally as an expressible nucleic acid or vector construct,(c) repression or activation of gene expression by use of CRISPR/CAS9 orCRISPR interference or interfering RNA, interfering mRNA or interferingaptamer, or siRNA or siRNA interference or a zinc finger transcriptionfactor or a zinc finger nuclease or a transcription activator-likeeffector nucleases (TALEN).

Where modifying the expression of the one or more genes comprises use ofan interfering RNA (RNAi), suitable RNAi include RNAi that decreases orincreases the level of a gene product, i.e. targets the one or moregenes. For example, an RNAi can be an shRNA or siRNA. A “smallinterfering” or “short interfering RNA” or siRNA is a RNA duplex ofnucleotides that is targeted to a gene interest or the one or moregenes. An “RNA duplex” refers to the structure formed by thecomplementary pairing between two regions of a RNA molecule. siRNA is“targeted” to a gene in that the nucleotide sequence of the duplexportion of the siRNA is complementary to a nucleotide sequence of thetargeted gene. In some embodiments, the length of the duplex of siRNAsis less than 30 nucleotides. In some embodiments, the duplex can be 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11or 10 nucleotides in length. In some embodiments, the length of theduplex is 19-25 nucleotides in length. The RNA duplex portion of thesiRNA can be part of a hairpin structure. In addition to the duplexportion, the hairpin structure may contain a loop portion positionedbetween the two sequences that form the duplex. The loop can vary inlength. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13nucleotides in length. The hairpin structure can also contain 3′ or 5′overhang portions. In some embodiments, the overhang is a 3′ or a 5′overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. A “short hairpinRNA,” or shRNA, is a polynucleotide construct that can be made toexpress an interfering RNA such as siRNA.

In some embodiments the vector contains one or more of a promotersequence, a directional cloning site, an epitope tag, a polyadenylationsequence, and antibiotic resistance gene. In some embodiments thepromoter sequence is Human cytomegalovirus immediate early promoter, thedirectional cloning site is TOPO, the epitope tag is V5 for detectionusing anti-V5 antibodies, the polyadenylation sequence is from HerpesSimplex Virus thymidine kinase, and antibiotic resistance gene isBlasticidin.

In some embodiments the cell growth and/or productivity inhibitor isselected from the group of consisting of: 3-(4-hydroxyphenyl)lactate(HPLA), 4-hydroxyphenylpyruvate, phenyllactate (PLA), indole carboxylate(indole-3-carboxylate), indole lactate (indole-3-lactate),2-hydroxybutyric acid, homocysteine, isovalerate, 2-methylbutyrate,isobutyrate, butyrate, formate.

In some embodiments the cell metabolic pathway or pathways synthesise3-(4-hydroxyphenyl)lactate (HPLA), 4-hydroxyphenylpyruvate,phenyllactate (PLA), indole carboxylate (indole-3-carboxylate), indolelactate (indole-3-lactate), 2-hydroxybutyric acid, homocysteine,isovalerate, 2-methylbutyrate, isobutyrate, butyrate, formate, ormetabolites thereof or metabolic intermediates thereof, or metabolite ofthe metabolic intermediate.

In some embodiments the cell metabolic pathway is the leucine pathwayand/or the isoleucine pathway and/or the valine pathway or thephenylalanine/tyrosine pathway or the acetoacetate/fumerate. In someembodiments the cell metabolic pathway is the leucine pathway or thephenylalanine/tyrosine pathway. In some embodiments the cell metabolicpathway is the leucine pathway and the isoleucine pathway and the valinepathway and/or the phenylalanine/tyrosine pathway.

In some embodiments the gene modified encodes an enzyme that catalysesthe synthesis of 3-(4-hydroxyphenyl)lactate (HPLA),4-hydroxyphenylpyruvate, phenyllactate (PLA), indole carboxylate(indole-3-carboxylate), indole lactate (indole-3-lactate),2-hydroxybutyric acid, homocysteine, isovalerate, 2-methylbutyrate,isobutyrate, butyrate, formate, or metabolites thereof or metabolicintermediates thereof, or metabolite of the metabolic intermediate.

In some embodiments the gene modified encodes an enzyme that catalysesthe synthesis of 4-hydroxyphenylpyruvate or phenyllactate (PLA) ormetabolites thereof or metabolic intermediates thereof, or metabolite ofthe metabolic intermediate.

In some embodiments the gene modified encodes an enzyme that catalysesthe synthesis of s isovalerate, 2-methylbutyrate, isobutyrate, orbutyrate, or metabolites thereof or metabolic intermediates thereof, ormetabolite of the metabolic intermediate.

In some embodiments the one or genes modified is selected from; PCDB1,QDPR, Pah, Mif, Got1, Got2, Nup62-il4i1, Hpd, Hgd, Gstz1, Fah.

In some embodiments the one or more genes modified is selected from;Bcat1, Bcat2, Bckdha/b, Dbt/Dld, Ivd, Acadm, Mccc1, Mccc2, Auh, Hmgcl,Fasn.

In some embodiments the one or genes modified is selected from; PCDB1,QDPR, Pah, Mif, Got1, Got2, Nup62-i14i1, Hpd, Hgd, Gstz1, Fah, Bcat1,Bcat2, Bckdha/b, Dbt/Dld, Ivd, Acadm, Mccc1, Mccc2, Auh, Hmgcl, Fasn,Fasn.

In some embodiments the one or more genes modified is selected from;Hpd, Hgd and Pah, PCDB1, QDPR.

In some embodiments the one or more genes modified is selected from,Bcat1, Bcat2, Auh, Mccc1, Mccc2, Ivd.

In some embodiments the one or more genes modified is selected from,Bcat1, Bcat2, Auh, Mccc1, Mccc2, Ivd, Hpd, Hgd and Pah, PCDB1, QDPR.

In some embodiments the gene is modified to increase or decrease geneexpression.

In some embodiments the gene is modified to decrease gene expression.

In some embodiments the modification (a) increases gene expression (b)increases gene expression except wherein the gene is Bcat1 and/or Bcat2,when modification decreases gene expression.

According to some embodiments there is provided a cell comprising one ormore modified genes which reduces the level of synthesis of growthand/or productivity inhibitors by the cell. In some embodiments the cellcomprises one or more modified genes selected from Bcat1, Bcat2, Auh,Mccc1/2, Ivd, Hpd, Hgd and Pah, PCDB1, QDPR, wherein the modificationincreases or decreases the gene expression, in some embodimentsincreases it, in some embodiments the gene expression of Bcat1 and/orBcat2 is reduced, in some embodiments the level of synthesis of growthand/or productivity inhibitors by the cell is also reduced.

In some embodiments the one or more genes modified is Auh, or is Bcat1,or is Bcat2, or is Bcat1 and Bcat2, or is one or more of Mccc1, Mccc2and Ivd, for example is Mccc1 or Mccc2 or Ivd optionally in combinationwith Auh and/or Bcat1 and/or Bcat2, or is Auh and one or more Mccc1,Mccc2 and Ivd, or is Auh, Mccc1, Mccc2 and Ivd, optionally incombination with Bcat1 and/or Bcat2. In some embodiments of thepreceding embodiments the one or more genes is modified to increase geneexpression, in some embodiments by mutation of the gene, in someembodiments by introduction of a copy of the wild type gene into thecell optionally as an expressible vector. In some embodiments Auh geneexpression can be increased by introduction of a copy of the wild typegene, in some embodiments by mutation, alternatively by both. In someembodiments Mccc1, Mccc2, Ivd gene expression can be increased bymutation, in some embodiments by introduction of a copy of the wild typegene, alternatively by both. In some embodiments Auh gene expression isincreased by introduction of a copy of the wild type gene and/or Mccc1,Mccc2, and Ivd gene expression is increased by mutation. In someembodiments the gene expression of Bcat1 and/or Bcat2 is reduced, eitherby gene knockdown or knockout, in some embodiments Bcat1 and/or Bcat2knockdown is to less than or equal to any of, 90, 80, 70, 60, 50, 45,40, 35, 30, 25, 20, 15, 10, 5, 2.5 percent level of Bcat1 and/or Bcat2expression or activity compared to unmodified cells. In someembodiments, the one or more modified gene(s) selected from (a) Pah,PCBD1, QDPR, Mif, Got1, Got2, Nup62-il4i1, Hpd, Hgd, Gstz1, Fah, Bcat1,Bcat2, Bckdha/b, Dbt/Dld, Ivd, Acadm, Mccc1, Mccc2, Auh, Hmgcl, Fasn,Fasn, (b) Pah, PCBD1, QDPR, Mif, Got1, Got2, Nup62-i14i1, Hpd, Hgd,Gstz1, Fah, (c) Bcat1, Bcat2, Bckdha/b, Dbt/Dld, Ivd, Acadm, Mccc1,Mccc2, Auh, Hmgcl, Fasn, Fasn, (d) Bcat1, Bcat2, Auh, Mccc1, Mccc2, Ivd,Hpd, Hgd and Pah, PCDB1, QDPR, wherein the modification increases ordecreases gene expression.

In some embodiments the one or more genes modified is Pah and/or PCDB1and/or QDPR, or is one or more of Hpd and Hgd, for example Hpd, or Hgdoptionally in combination with Pah and/or PCDB1 and/or QDPR, or is Pahand one or more of Hpd and Hgd or, is Pah, Hpd and Hgd. In someembodiments the one or more genes modified is selected from; (i) PCDB1,(ii) Pah, (iii) QDPR, (iv) PCBD1 and QDPR, (v) PCBD1 and Pah, (vi) Pahand QDPR, (vii) PCDB1 and Pah, and QDPR, (viii) any of (i) to (vii) andHpd and/or Hgd. In some embodiments of the preceding embodiments the oneor more genes is modified to increase gene expression. In someembodiments any one or more of Pah, PCDB1, QDPR, Hpd, Hgd geneexpression can be increased by introduction of a copy of the wild typegene, in some embodiments by mutation, alternatively by both. In someembodiments Pah, PCDB1, QDPR, Hpd and Hgd gene expression is increasedby introduction of a copy of the wild type gene. In some embodiments Hpdand Hgd gene expression is increased by introduction of a copy of thewild type gene. In some embodiments Pah, PCDB1 and/or QDPR geneexpression is increased by introduction of a copy of the wild type gene.

In some embodiments, the one or more genes modified is Auh, Mccc1,Mccc2, Ivd, Pah, Hpd and Hgd. In some embodiments, the one or more genesmodified is Auh, Mccc1, Mccc2, Ivd, Pah, PCDB1, Hpd and Hgd andoptionally QDPR. In some embodiments, the one or more genes modified isBcat1 and/or Bcat2, Pah, PCDB1, Hpd and Hgd and optionally QDPR. In someembodiments of the preceding embodiments the one or more genes ismodified to increase gene expression by way of the relevant method ofintroduction of a copy of the wild type gene, by mutation, alternativelyby both. In some embodiments Auh gene expression is increased byintroduction of a copy of the wild type gene and Mccc1, Mccc2, Ivd, Pah,PCDB1, QDPR, Hpd and Hgd gene expression is increased by mutation.

The present invention further provides a cell obtained or obtainable bythe method of producing cells. In some embodiments the cell comprisesone or more modified genes which reduces the level of synthesis ofgrowth and/or productivity inhibitors by the cell. In some embodimentsthe cell comprises one or more modified genes selected from Bcat1,Bcat2, Auh, Mccc1, Mccc2, Ivd, Hpd, Hgd and Pah, PCDB1, QDPR, whereinthe modification increases or decreases the gene expression. In someembodiments gene expression is increased. In some embodiments the levelof synthesis of growth and/or productivity inhibitors by the cell isalso reduced.

In some embodiments of the cell obtained by the method of producingcells the one or more genes modified is Auh, or is Bcat1, or is Bcat2,or is Bcat1 and Bcat2, or is one or more of Mccc1, Mccc2 and Ivd, forexample is Mccc1 or Mccc2 or Ivd optionally in combination with Auhand/or Bcat1 and/or Bcat2, or is Auh and one or more Mccc1, Mccc2 andIvd, or is Auh, Mccc1, Mccc2 and Ivd, optionally in combination withBcat1 and/or Bcat2. In some embodiments of the preceding embodiments theone or more genes is modified to increase gene expression, in someembodiments by mutation of the gene, in some embodiments by introductionof a copy of the wild type gene into the cell optionally as anexpressible vector. In some embodiments Auh gene expression can beincreased by introduction of a copy of the wild type gene, in someembodiments by mutation, alternatively by both. In some embodimentsMccc1, Mccc2, Ivd gene expression can be increased by mutation, in someembodiments by introduction of a copy of the wild type gene,alternatively by both. In some embodiments Auh gene expression isincreased by introduction of a copy of the wild type gene and/or Mccc1,Mccc2, and Ivd gene expression is increased by mutation. In someembodiments the gene expression of Bcat1 and/or Bcat2 is reduced, eitherby gene knockdown or knockout, in some embodiments Bcat1 and/or Bcat2knockdown is to less than or equal to any of, 90, 80, 70, 60, 50, 45,40, 35, 30, 25, 20, 15, 10, 5, 2.5 percent level of Bcat1 and/or Bcat2expression or activity compared to unmodified cells.

In some embodiments the one or more genes modified is Pah, and/or PCDB1and/or QDPR, or is one or more of Hpd and Hgd, for example Hpd, or Hgdoptionally in combination with Pah, and/or PCDB1 and/or QDPR, or is Pahand one or more of Hpd and Hgd. or, is Pah, Hpd and Hgd. In someembodiments the one or more genes modified is selected from; (i) PCDB1,(ii) Pah, (iii) QDPR, (iv) PCBD1 and QDPR, (v) PCBD1 and Pah, (vi) Pahand QDPR, (vii) PCDB1 and Pah, and QDPR, (viii) any of (i) to (vii) andHpd and/or Hgd. In some embodiments of the preceding embodiments the oneor more genes is modified to increase gene expression. In someembodiments Pah, PCDB1, QDPR, Hpd, Hgd gene expression can be increasedby introduction of a copy of the wild type gene, in some embodiments bymutation, alternatively by both. In some embodiments Pah, PCDB1, QDPR,Hpd and Hgd gene expression is increased by introduction of a copy ofthe wild type gene. In some embodiments Hpd and Hgd gene expression isincreased by introduction of a copy of the wild type gene. In someembodiments Pah, PCDB1, QDPR, Hpd and Hgd gene expression is increasedby introduction of a copy of the wild type gene.

In some embodiments the one or more genes modified is Auh, Mccc1, Mccc2,Ivd, Pah, Hpd and Hgd. In some embodiments, the one or more genesmodified is Auh, Mccc1, Mccc2, Ivd, Pah, PCDB1, Hpd and Hgd andoptionally QDPR. In some embodiments, the one or more genes modified isBcat1 and/or Bcat2, Pah, PCDB1, Hpd and Hgd and optionally QDPR. In someembodiments of the preceding embodiments the one or more genes ismodified to increase gene expression by way of the relevant method ofintroduction of a copy of the wild type gene, by mutation, alternativelyby both. In some embodiments Auh gene expression is increased byintroduction of a copy of the wild type gene and Mccc1, Mccc2, Ivd, Pah,PCDB1, QDPR, Hpd and Hgd gene expression is increased by mutation.

In some embodiments the one or more genes modified is selected fromBcat1, Bcat2, Auh, Mccc1, Mccc2, Ivd, Hpd, Hgd, PCBD1, QDPR and Pah,wherein the modification increases the gene expression of one or more ofAuh, Mccc1, Mccc2, Ivd, Hpd, Hgd, PCBD1, QDPR and Pah and/or reduces thegene expression of Bcat1 or Bcat2 and/or reduces the level of synthesisof growth and/or productivity inhibitors by the cell.

In some embodiments the one or more genes modified is, (i) Auh, (ii)Bcat1, (iii) Bcat2, (iv) one or more of Mccc1, Mccc2 and Ivd, (v) Bcat1and/or Bcat2 and/or Auh and one or more Mccc1, Mccc2 and Ivd, (vi) Pah,(vii) PCBD1, (viii) PCBD1 and/or QDPR, (ix) one or more of Hpd and Hgd,(x) Pah and/or PCBD1 and one or more of Hpd and Hgd, (xi) Pah and PCBD1,and optionally QDPR, (xii) Pah, PCBD1, Hpd and Hgd, and optionally QDPR,(xiii) Bcat1 and/or Bcat2, Auh, Mccc1, Mccc2 and Ivd or, (xiv) Bcat1and/or Bcat2, Auh, Mccc1, Mccc2, Ivd, Pah, PCBD1, Hpd, Hgd andoptionally QDPR, (xv) Bcat1 and/or Bcat2. In some embodiments themodification (a) increases gene expression (b) increases gene expressionexcept wherein the gene is Bcat1 and/or Bcat2, when modificationdecreases gene expression, optionally Bcat1 and/or Bcat2 knockdown is toless than or equal to any of, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25,20, 15, 10, 5, 2.5 percent level of Bcat1 and/or Bcat2 expression oractivity compared to unmodified cells. In one embodiment the cellsexpress a recombinant protein or heterologous recombinant protein.

Cells

Any cell susceptible to cell culture may be utilized in accordance withthe present invention. In some embodiments, the cell is a mammaliancell. Non-limiting examples of mammalian cells that may be used inaccordance with the present invention include BALB/c mouse myeloma line(NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell,Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells +/−DHFR (CHO, Urlaub and Chasin, Proc. Natl.Acad. Sci. USA, 77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1 587); humancervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al.,Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2). In some preferred embodiment, the cellsare CHO cells. In some preferred embodiments, the cells are GS-cells.

Additionally, any number of commercially and non-commercially availablehybridoma cell lines may be utilized in accordance with the presentinvention. The term “hybridoma” as used herein refers to a cell orprogeny of a cell resulting from fusion of an immortalized cell and anantibody-producing cell. Such a resulting hybridoma is an immortalizedcell that produces antibodies. Individual cells used to create thehybridoma can be from any mammalian source, including, but not limitedto, rat, pig, rabbit, sheep, pig, goat, and human. In some embodiments,a hybridoma is a trioma cell line, which results when progeny ofheterohybrid myeloma fusions, which are the product of a fusion betweenhuman cells and a murine myeloma cell line, are subsequently fused witha plasma cell. In some embodiments, a hybridoma is any immortalizedhybrid cell line that produces antibodies such as, for example,quadromas (See, e.g., Milstein et al., Nature, 537:3053, 1983). Oneskilled in the art will appreciate that hybridoma cell lines might havedifferent nutrition requirements and/or might require different cultureconditions for optimal growth, and will be able to modify conditions asneeded.

Methods Comprising Identifying and/or Measuring the MetaboliteConcentration in the Cell Culture Medium or in Cells (Cell PelletSample)

Metabolites can be identified and/or the concentration of metabolitescan be measured by any method known to the skilled person, including offline and on line measurement methods, such measurements also constitutemetabolomic analysis or metabolomic measurement. Applied to the cellculture medium or to samples of the cells for instance the cell pellet

The identification and/or concentration of metabolites can be measuredonce or several times during the cell culture. In some embodiments, themetabolite concentration is measured continuously, intermittently, every30 min, every hour, every two hours, twice a day, daily, or every twodays. In a preferred embodiment the identification and/or concentrationof metabolite is measured daily.

An off line measurement method as used herein refers to a method wherethe measurement of a parameter such as a concentration is not automatedand integrated to the cell culture method. For example, a measurementmethod where a sample is manually taken from the cell culture medium sothat a specific concentration can be measured in said sample isconsidered as an off line measurement method.

Online measurement methods as used herein refer to methods where themeasurement of a parameter, such as a concentration, is automated andintegrated to the cell culture method.

For example, a method using the Raman spectroscopy as disclosed inExample 7 is an on-line measurement method. Alternatively, the use ofHigh Performance Liquid Chromatography (HPLC) or Ultra PerformanceLiquid Chromatography (UPLC) based technology with an auto-sampler thatdraws samples from reactor and transfers them to the equipment in aprogrammed manner is an online measurement method.

The identification and/or concentration of metabolites can be measuredby any method known to the skilled person. Preferred methods to identifyand/or measure the concentration of metabolites in online or offlinemethods include for example Liquid Chromatography such asHigh-Performance Liquid Chromatography (HPLC), Ultra Performance LiquidChromatography (UPLC) or Liquid Chromatography-Mass Spectrometry (LCMS),Nuclear Magnetic Resonance (NMR) or Gas Chromatography-Mass Spectrometry(GCMS).

In some embodiments, the identification and/or concentration ofmetabolite is measured off line by taking a sample of the cell culturemedium and measuring the concentration of said at least one metabolitein said sample. In some embodiments, the identification and/orconcentration of metabolites is measured as disclosed in Example 2. Apreferred method to measure the identification and/or concentration ofmetabolites in an offline method is LCMS.

In some embodiments, the identification and/or concentration ofmetabolite is measured on-line. In some embodiments, the identificationand/or concentration of metabolite is measured online using Ramanspectroscopy. In some embodiments, the identification and/orconcentration of metabolite is measured on-line using Raman spectroscopyas disclosed in Example 7. In some embodiments, the identificationand/or concentration of metabolite is measured online using HPLC or UPLCbased technology with an auto-sampler that draws samples from reactorand transfers them to the equipment in a programmed manner.Identification of a metabolite includes determining the presence and/oridentity of the metabolite.

Improvement of Cell Growth and Productivity

In some embodiments of the above described methods, cell growth and/orproductivity are increased as compared to a control culture, saidcontrol culture being identical except that it does not comprise step(ii) and/or (iii) and/or does not comprise the modified cells or thecells produced by the method of producing cells. i.e. the cells areunmodified.

In some embodiments of the above described methods, the method of theinvention is a method for improving cell growth. In some embodiments,the method of the invention is a method for improving cell growth inhigh density cell culture at high cell density.

High cell density as used herein refers to cell density above 1×10⁶cells/mL, 5×10⁶ cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL, 1×10⁸ cells/mLor 5×10⁸ cells/mL, preferably above 1×10⁷ cells/mL, more preferablyabove 5×10⁷ cells/mL.

In some embodiments, the above described methods are for improving cellgrowth in a cell culture where cell density is above 1×10⁶ cells/mL,5×10⁶ cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL, 1×10⁸ cells/mL or 5×10⁸cells/mL.

In some embodiments, the methods are for improving cell growth in a cellculture where maximum cell density is above 1×10⁶ cells/mL, 5×10⁶cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL, 1×10⁸ cells/mL or 5×10⁸cells/mL.

In some embodiments, cell growth is determined by viable cell density(VCD), maximum viable cell density, or Integrated viable cell count(IVCC). In some embodiments, cell growth is determined by maximum viablecell density.

The term “viable cell density” as used herein refers to the number ofcells present in a given volume of medium. Viable cell density can bemeasured by any method known to the skilled person. Preferably, Viablecell density is measured using an automated cell counter such asBioprofile Flex®. The term maximum cell density as used herein refers tothe maximum cell density achieved during the cell culture.

The term “cell viability” as used herein refers to the ability of cellsin culture to survive under a given set of culture conditions orexperimental variations. Those of ordinary skill in the art willappreciate that one of many methods for determining cell viability areencompassed in this invention. For example, one may use a dye (e.g.,trypan blue) that does not pass through the membrane of a living cell,but can pass through the disrupted membrane of a dead or dying cell inorder to determine cell viability.

The term “Integrated viable cell count (IVCC)” as used herein refers toas the area under the viable cell density (VCD) curve. IVCC can becalculated using the following formula:IVCC_(t+1)=IVCC_(t)+(VCD_(t)+VCD_(t+1))*(Δt)/2where Δt is the time difference between t and t+1 time points.IVCC_(t=0) can be assumed negligible. VCD_(t) and VCD_(t+1) are viablecell densities at t and t+1 time points.

The term “titer” as used herein refers, for example, to the total amountof recombinantly expressed protein produced by a cell culture in a givenamount of medium volume. Titer is typically expressed in units of gramsof protein per liter of medium.

In some embodiments of the above described methods, cell growth isincreased by at least 5%, 10%, 15%, 20% or 25% as compared to thecontrol culture. In some embodiments, cell growth is increased by atleast 10% as compared to the control culture. In some embodiments, cellgrowth is increased by at least 20% as compared to the control culture.

In some embodiments of the above described methods, the productivity isdetermined by titer and/or volumetric productivity.

The term “titer” as used herein refers, for example, to the total amountof recombinantly expressed protein produced by a cell culture in a givenamount of medium volume. Titer is typically expressed in units of gramsof protein per liter of medium.

In some embodiments of the above described methods, the productivity isdetermined by titer. In some embodiments, the productivity is increasedby at least 5%, 10%, 15%, 20% or 25% as compared to the control culture.In some embodiments, the productivity is increased by at least 10% ascompared to a control culture. In some embodiments, the productivity isincreased by at least 20% as compared to a control culture.

In some embodiments of the above described methods, the maximum celldensity of the cell culture is greater than 1×10⁶ cells/mL, 5×10⁶cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL, 1×10⁸ cells/mL or 5×10⁸cells/mL. In some embodiments, the maximum cell density of the cellculture is greater than 5×10⁶ cells/mL. In some embodiments, the maximumcell density of the cell culture is greater than 1×10⁸ cells/mL.

Cell Culture Media

The terms “medium”, “cell culture medium” and “culture medium” as usedherein refer to a solution containing components or nutrients whichnourish growing mammalian cells. Typically, the nutrients includeessential and non-essential amino acids, vitamins, energy sources,lipids, and trace elements required by the cell for minimal growthand/or survival. Such a solution may also contain further nutrients orsupplementary components that enhance growth and/or survival above theminimal rate, including, but not limited to, hormones and/or othergrowth factors, particular ions (such as sodium, chloride, calcium,magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), inorganic compounds present at high finalconcentrations (e.g., iron), amino acids, lipids, and/or glucose orother energy source. In some embodiments, a medium is advantageouslyformulated to a pH and salt concentration optimal for cell survival andproliferation. In some embodiments, a medium is a feed medium that isadded after the beginning of the cell culture.

A wide variety of mammalian growth media may be used in accordance withthe present invention. In some embodiments, cells may be grown in one ofa variety of chemically defined media, wherein the components of themedia are both known and controlled. In some embodiments, cells may begrown in a complex medium, in which not all components of the medium areknown and/or controlled. Chemically defined growth media for mammaliancell culture have been extensively developed and published over the lastseveral decades. All components of defined media are well characterized,and so defined media do not contain complex additives such as serum orhydrolysates. Early media formulations were developed to permit cellgrowth and maintenance of viability with little or no concern forprotein production. More recently, media formulations have beendeveloped with the express purpose of supporting highly productiverecombinant protein producing cell cultures. Such media are preferredfor use in the method of the invention. Such media generally compriseshigh amounts of nutrients and in particular of amino acids to supportthe growth and/or the maintenance of cells at high density. Ifnecessary, these media can be modified by the skilled person for use inthe method of the invention. For example, the skilled person maydecrease the amount of phenylalanine, tyrosine, tryptophan and/ormethionine in these media for their use as base media or feed media in amethod as disclosed herein.

Not all components of complex media are well characterized, and socomplex media may contain additives such as simple and/or complex carbonsources, simple and/or complex nitrogen sources, and serum, among otherthings. In some embodiments, complex media suitable for the presentinvention contains additives such as hydrolysates in addition to othercomponents of defined medium as described herein.

In some embodiments, defined media typically includes roughly fiftychemical entities or components at known concentrations in water. Mostof them also contain one or more well-characterized proteins such asinsulin, IGF-1, transferrin or BSA, but others require no proteincomponents and so are referred to as protein-free defined media. Typicalchemical components of the media fall into five broad categories: aminoacids, vitamins, inorganic salts, trace elements, and a miscellaneouscategory that defies neat categorization.

Cell culture medium may be optionally supplemented with supplementarycomponents. The term “supplementary components” as used herein refers tocomponents that enhance growth and/or survival above the minimal rate,including, but not limited to, hormones and/or other growth factors,particular ions (such as sodium, chloride, calcium, magnesium, andphosphate), buffers, vitamins, nucleosides or nucleotides, traceelements (inorganic compounds usually present at very low finalconcentrations), amino acids, lipids, and/or glucose or other energysource. In some embodiments, supplementary components may be added tothe initial cell culture. In some embodiments, supplementary componentsmay be added after the beginning of the cell culture. Typically,components which are trace elements refer to a variety of inorganicsalts included at micromolar or lower levels. For example, commonlyincluded trace elements are zinc, selenium, copper, and others. In someembodiments, iron (ferrous or ferric salts) can be included as a traceelement in the initial cell culture medium at micromolar concentrations.Manganese is also frequently included among the trace elements as adivalent cation (MnCl₂ or MnSO₄) in a range of nanomolar to micromolarconcentrations. Numerous less common trace elements are usually added atnanomolar concentrations.

In some embodiments, the medium used in the methods of the invention isa medium suitable for supporting high cell density, such as for example1×10⁶ cells/mL, 5×10⁶ cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL, 1×10⁸cells/mL or 5×10⁸ cells/mL, in a cell culture. In some embodiments, thecell culture is a mammalian cell fed-batch culture, preferably a CHOcells fed-batch culture.

In some embodiments, the cell culture medium comprises phenylalanine ata concentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tyrosine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM. In someembodiments, the cell culture medium comprises no tyrosine. In someembodiments, the cell culture medium is HiPDOG medium, medium used inthe HipDOG process, but comprising no tyrosine. In some embodiments thecell culture medium comprises no tyrosine wherein a HiPDOG culture orprocess is used which comprises no tyrosine.

In some embodiments, the cell culture medium comprises tryptophan at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises methionine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises leucine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises isoleucine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises valine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises serine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises threonine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises glycine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises two ofphenylalanine, tyrosine, tryptophan, methionine, leucine, isoleucine,valine, serine, threonine and glycine at a concentration below 2 mM,below 1 mM, between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine andtyrosine at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine andtryptophan at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine andmethionine at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tyrosine andtryptophan at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tyrosine andmethionine at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tryptophan andmethionine at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises three ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine and glycine at a concentration below 2 mM, below 1 mM, between0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tyrosine and tryptophan at a concentration below 2 mM, below 1 mM,between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM orbetween 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tyrosine and methionine at a concentration below 2 mM, below 1 mM,between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM orbetween 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tryptophan and methionine at a concentration below 2 mM, below 1 mM,between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM orbetween 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tyrosine,tryptophan and methionine at a concentration below 2 mM, below 1 mM,between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM orbetween 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises four ofphenylalanine, tyrosine, tryptophan, methionine, leucine, isoleucine,valine, serine, threonine and glycine at a concentration below 2 mM,below 1 mM, between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tyrosine, tryptophan and methionine at a concentration below 2 mM, below1 mM, between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mMor between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises five ofphenylalanine, tyrosine, tryptophan, methionine, leucine, isoleucine,valine, serine, threonine and glycine at a concentration below 2 mM,below 1 mM, between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises six ofphenylalanine, tyrosine, tryptophan, methionine, leucine, isoleucine,valine, serine, threonine and glycine at a concentration below 2 mM,below 1 mM, between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises seven ofphenylalanine, tyrosine, tryptophan, methionine, leucine, isoleucine,valine, serine, threonine and glycine at a concentration below 2 mM,below 1 mM, between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tyrosine, tryptophan, methionine, leucine, isoleucine, valine, serine,threonine and glycine at a concentration below 2 mM, below 1 mM, between0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5to 1 mM.

In some embodiments, the cell culture medium further comprises at least1, 2, 3, 4, 5, 6, 7, 8, or 9, of glycine, proline, serine, threonine,lysine, arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2mM.

In some embodiments, the cell culture medium further comprises at least5 of glycine, proline, serine, threonine, lysine, arginine, histidine,aspartate, glutamate and asparagine at a concentration above 2 mM, 3 mM,4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM.

In some embodiments, the cell culture medium further comprises glycine,proline, serine, threonine, lysine, arginine, histidine, aspartate,glutamate and asparagine at a concentration above 2 mM, 3 mM, 4 mM, 5mM, 10 mM, 15 mM, preferably 2 mM. In some embodiments, the cell culturemedium further comprises at least 1, 2, 3, 4, 5, 6, or 7, of proline,lysine, arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2mM.

In some embodiments, the cell culture medium further comprises at least5 of proline, lysine, arginine, histidine, aspartate, glutamate andasparagine at a concentration above 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15mM, preferably 2 mM.

In some embodiments, the cell culture medium further comprises proline,lysine, arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2mM.

In some embodiments, the cell culture medium comprises serine at aconcentration above 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably10 mM.

In some embodiments, the cell culture medium comprises cysteine at aconcentration above 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably10 mM.

In some embodiments, the above cell culture medium is for use in amethod as disclosed herein. In some embodiments, the above cell culturemedium is used in a method as disclosed herein as a base media. In someembodiments, the above cell culture medium is used a method as disclosedherein as a feed media.

Expression of Proteins

As noted above, in many instances the cells will be selected orengineered to produce high levels of desired products (e.g., recombinantprotein or antibody). Often, cells will be manipulated by the hand ofman to produce high levels of recombinant protein, for example byintroduction of a gene encoding the protein of interest and/or byintroduction of genetic control elements that regulate expression ofthat gene (whether endogenous or introduced).

Certain proteins may have detrimental effects on cell growth, cellviability or some other characteristic of the cells that ultimatelylimits production of the protein of interest in some way. Even amongst apopulation of cells of one particular type engineered to express aspecific protein, variability within the cellular population exists suchthat certain individual cells will grow better, produce more protein ofinterest, or produce a protein with higher activity levels (e.g.,enzymatic activity). In certain embodiments of the invention, a cellline is empirically selected by the practitioner for robust growth underthe particular conditions chosen for culturing the cells. In someembodiments, individual cells engineered to express a particular proteinare chosen for large-scale production based on cell growth, final celldensity, percent cell viability, titer of the expressed protein or anycombination of these or any other conditions deemed important by thepractitioner.

Any protein that is expressible in a host cell may be produced inaccordance with the present teachings and may be produced according tothe methods of the invention or by the cells of the invention. The term“host cell” as used herein refers to a cell that is manipulatedaccording to the present invention to produce a protein of interest asdescribed herein. A protein may be expressed from a gene that isendogenous to the cell, or from a heterologous gene that is introducedinto the cell. A protein may be one that occurs in nature, or mayalternatively have a sequence that was engineered or selected by thehand of man.

Proteins that may desirably be expressed in accordance with the presentinvention will often be selected on the basis of an interesting oruseful biological or chemical activity. For example, the presentinvention may be employed to express any pharmaceutically orcommercially relevant enzyme, receptor, antibody, hormone, regulatoryfactor, antigen, binding agent, etc. In some embodiments, the proteinexpressed by cells in culture are selected from antibodies, or fragmentsthereof, nanobodies, single domain antibodies, glycoproteins,therapeutic proteins, growth factors, clotting factors, cytokines,fusion proteins, pharmaceutical drug substances, vaccines, enzymes, orSmall Modular ImmunoPharmaceuticals™ (SMIPs). One of ordinary skill inthe art will understand that any protein may be expressed in accordancewith the present invention and will be able to select the particularprotein to be produced based on his or her particular needs.

Antibodies

Given the large number of antibodies currently in use or underinvestigation as pharmaceutical or other commercial agents, productionof antibodies is of particular interest in accordance with the presentinvention. Antibodies are proteins that have the ability to specificallybind a particular antigen. Any antibody that can be expressed in a hostcell may be produced in accordance with the present invention and may beproduced according to the methods of the invention or by the cells ofthe invention. In some embodiments, the antibody to be expressed is amonoclonal antibody.

In some embodiments, the monoclonal antibody is a chimeric antibody. Achimeric antibody contains amino acid fragments that are derived frommore than one organism. Chimeric antibody molecules can include, forexample, an antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimeric antibodies have been described. See e.g., Morrison etal., Proc. Natl. Acad. Sci. U.S.A. 81, 6851 (1985); Takeda et al.,Nature 314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss etal., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B.

In some embodiments, the monoclonal antibody is a human antibodyderived, e.g., through the use of ribosome-display or phage-displaylibraries (see, e.g., Winter et al., U.S. Pat. No. 6,291,159 andKawasaki, U.S. Pat. No. 5,658,754) or the use of xenographic species inwhich the native antibody genes are inactivated and functionallyreplaced with human antibody genes, while leaving intact the othercomponents of the native immune system (see, e.g., Kucherlapati et al.,U.S. Pat. No. 6,657,103).

In some embodiments, the monoclonal antibody is a humanized antibody. Ahumanized antibody is a chimeric antibody wherein the large majority ofthe amino acid residues are derived from human antibodies, thusminimizing any potential immune reaction when delivered to a humansubject. In humanized antibodies, amino acid residues in thecomplementarity determining regions are replaced, at least in part, withresidues from a non-human species that confer a desired antigenspecificity or affinity. Such altered immunoglobulin molecules can bemade by any of several techniques known in the art, (e.g., Teng et al.,Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al.,Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92,3-16 (1982)), and are preferably made according to the teachings of PCTPublication WO92/06193 or EP 0239400, all of which are incorporatedherein by reference). Humanized antibodies can be commercially producedby, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex,Great Britain. For further reference, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992), all of which areincorporated herein by reference.

In some embodiments, the monoclonal, chimeric, or humanized antibodiesdescribed above may contain amino acid residues that do not naturallyoccur in any antibody in any species in nature. These foreign residuescan be utilized, for example, to confer novel or modified specificity,affinity or effector function on the monoclonal, chimeric or humanizedantibody. In some embodiments, the antibodies described above may beconjugated to drugs for systemic pharmacotherapy, such as toxins,low-molecular-weight cytotoxic drugs, biological response modifiers, andradionuclides (see e.g., Kunz et al., Calicheamicin derivative-carrierconjugates, US20040082764 A1).

In general, practitioners of the present invention will select a proteinof interest, and will know its precise amino acid sequence. Any givenprotein that is to be expressed in accordance with the present inventionmay have its own particular characteristics and may influence the celldensity or viability of the cultured cells, may be expressed at lowerlevels than another protein grown under identical culture conditions,and may have different biological activity depending on the exactculture conditions and steps performed. One of ordinary skill in the artwill be able to appropriately modify the steps and compositions used toproduce a particular protein according to the teachings of the presentinvention in order to optimize cell growth and the production and/oractivity level of any given expressed protein.

Introduction of Genes for the Expression of Proteins into Host Cells

Generally, a nucleic acid molecule introduced into the cell encodes theprotein desired to be expressed according to the present invention andmay be introduced and expressed according to the methods of theinvention or into and by the cells of the invention. Alternatively, anucleic acid molecule may encode a gene product that induces theexpression of the desired protein by the cell. For example, introducedgenetic material may encode a transcription factor that activatestranscription of an endogenous or heterologous protein. Alternatively oradditionally, an introduced nucleic acid molecule may increase thetranslation or stability of a protein expressed by the cell.

Methods suitable for introducing nucleic acids sufficient to achieveexpression of a protein of interest into mammalian host cells are knownin the art. See, for example, Gething et al., Nature, 293:620-625, 1981;Mantei et al., Nature, 281:40-46, 1979; Levinson et al. EP 117,060; andEP 117,058, each of which is incorporated herein by reference. Formammalian cells, common methods of introducing genetic material intomammalian cells include the calcium phosphate precipitation method ofGraham and van der Erb (Virology, 52:456-457, 1978) or theLipofectamine™ (Gibco BRL) Method of Hawley-Nelson (Focus 15:73, 1993).General aspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. Forvarious techniques for introducing genetic material into mammaliancells, see Keown et al., Methods in Enzymology, 1989, Keown et al.,Methods in Enzymology, 185:527-537, 1990, and Mansour et al., Nature,336:348-352, 1988. Additional methods suitable for introducing nucleicacids include electroporation, for example as employed using theGenePulser XCell™ electroporator by BioRad™.

In some embodiments, a nucleic acid to be introduced is in the form of anaked nucleic acid molecule. For example, the nucleic acid moleculeintroduced into a cell may consist only of the nucleic acid encoding theprotein and the necessary genetic control elements. Alternatively, anucleic acid encoding the protein (including the necessary regulatoryelements) may be contained within a plasmid vector. Non-limitingrepresentative examples of suitable vectors for expression of proteinsin mammalian cells include pCDNA1; pCD, see Okayama, et al. Mol. CellBiol. 5:1136-1142, 1985; pMClneo Poly-A, see Thomas, et al. Cell51:503-512, 1987; a baculovirus vector such as pAC 373 or pAC 610; CDM8,see Seed, B. Nature 329:840, 1987; and pMT2PC, see Kaufman, et al. EMBOJ. 6:187-195, 1987, each of which is incorporated herein by reference inits entirety. In some embodiments, a nucleic acid molecule to beintroduced into a cell is contained within a viral vector. For example,a nucleic acid encoding the protein may be inserted into the viralgenome (or a partial viral genome). Regulatory elements directing theexpression of the protein may be included with the nucleic acid insertedinto the viral genome (i.e., linked to the gene inserted into the viralgenome) or can be provided by the viral genome itself.

Naked DNA can be introduced into cells by forming a precipitatecontaining the DNA and calcium phosphate. Alternatively, naked DNA canalso be introduced into cells by forming a mixture of the DNA andDEAE-dextran and incubating the mixture with the cells or by incubatingthe cells and the DNA together in an appropriate buffer and subjectingthe cells to a high-voltage electric pulse (e.g., by electroporation). Afurther method for introducing naked DNA cells is by mixing the DNA witha liposome suspension containing cationic lipids. The DNA/liposomecomplex is then incubated with cells. Naked DNA can also be directlyinjected into cells by, for example, microinjection.

Alternatively, naked DNA can also be introduced into cells by complexingthe DNA to a cation, such as polylysine, which is coupled to a ligandfor a cell-surface receptor (see for example Wu, G. and Wu, C. H. J.Biol. Chem. 263:14621, 1988; Wilson et al. J. Biol. Chem. 267:963-967,1992; and U.S. Pat. No. 5,166,320, each of which is hereby incorporatedby reference in its entirety). Binding of the DNA-ligand complex to thereceptor facilitates uptake of the DNA by receptor-mediated endocytosis.

Use of viral vectors containing particular nucleic acid sequences, e.g.,a cDNA encoding a protein, is a common approach for introducing nucleicacid sequences into a cell. Infection of cells with a viral vector hasthe advantage that a large proportion of cells receive the nucleic acid,which can obviate the need for selection of cells which have receivedthe nucleic acid. Additionally, molecules encoded within the viralvector, e.g., by a cDNA contained in the viral vector, are generallyexpressed efficiently in cells that have taken up viral vector nucleicacid. Defective retroviruses are well characterized for use in genetransfer for gene therapy purposes (for a review see Miller, A. D. Blood76:271, 1990). A recombinant retrovirus can be constructed having anucleic acid encoding a protein of interest inserted into the retroviralgenome. Additionally, portions of the retroviral genome can be removedto render the retrovirus replication defective. Such a replicationdefective retrovirus is then packaged into virions which can be used toinfect a target cell through the use of a helper virus by standardtechniques.

The genome of an adenovirus can be manipulated such that it encodes andexpresses a protein of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See, forexample, Berkner et al. BioTechniques 6:616, 1988; Rosenfeld et al.Science 252:431-434, 1991; and Rosenfeld et al. Cell 68:143-155, 1992.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) areknown to those skilled in the art. Recombinant adenoviruses areadvantageous in that they do not require dividing cells to be effectivegene delivery vehicles and can be used to infect a wide variety of celltypes, including airway epithelium (Rosenfeld et al., 1992, citedsupra), endothelial cells (Lemarchand et al., Proc. Natl. Acad. Sci. USA89:6482-6486, 1992), hepatocytes (Herz and Gerard, Proc. Natl. Acad.Sci. USA 90:2812-2816, 1993) and muscle cells (Quantin et al., Proc.Natl. Acad. Sci. USA 89:2581-2584, 1992). Additionally, introducedadenoviral DNA (and foreign DNA contained therein) is not integratedinto the genome of a host cell but remains episomal, thereby avoidingpotential problems that can occur as a result of insertional mutagenesisin situations where introduced DNA becomes integrated into the hostgenome (e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham, J. Virol. 57:267, 1986). Most replication-defectiveadenoviral vectors currently in use are deleted for all or parts of theviral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material.

Adeno-associated virus (AAV) is a naturally occurring defective virusthat requires another virus, such as an adenovirus or a herpes virus, asa helper virus for efficient replication and a productive life cycle.(For a review see Muzyczka et al. Curr. Topics in Micro. and Immunol.,158:97-129, 1992). It is also one of the few viruses that may integrateits DNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al., Am. J. Respir. Cell. Mol.Biol. 7:349-356, 1992; Samulski et al., J. Virol. 63:3822-3828, 1989;and McLaughlin et al., J. Virol. 62:1963-1973, 1989). Vectors containingas little as 300 base pairs of AAV can be packaged and can integrate.Space for exogenous DNA is limited to about 4.5 kb. An AAV vector suchas that described in Tratschin et al. (Mol. Cell. Biol. 5:3251-3260,1985) can be used to introduce DNA into cells. A variety of nucleicacids have been introduced into different cell types using AAV vectors(see for example Hermonat et al., Proc. Natl. Acad. Sci. USA81:6466-6470, 1984; Tratschin et al., Mol. Cell. Biol. 4:2072-2081,1985; Wondisford et al., Mol. Endocrinol. 2:32-39, 1988; Tratschin etal., J. Virol. 51:611-619, 1984; and Flotte et al., J. Biol. Chem.268:3781-3790, 1993).

When the method used to introduce nucleic acid molecules into apopulation of cells results in modification of a large proportion of thecells and efficient expression of the protein by the cells, the modifiedpopulation of cells may be used without further isolation or subcloningof individual cells within the population. That is, there may besufficient production of the protein by the population of cells suchthat no further cell isolation is needed and the population can beimmediately be used to seed a cell culture for the production of theprotein. Alternatively, it may be desirable to isolate and expand ahomogenous population of cells from a few cells or a single cell thatefficiently produce(s) the protein. Alternative to introducing a nucleicacid molecule into a cell that encodes a protein of interest, theintroduced nucleic acid may encode another polypeptide or protein thatinduces or increases the level of expression of the protein producedendogenously by a cell. For example, a cell may be capable of expressinga particular protein but may fail to do so without additional treatmentof the cell. Similarly, the cell may express insufficient amounts of theprotein for the desired purpose. Thus, an agent that stimulatesexpression of the protein of interest can be used to induce or increaseexpression of that protein by the cell. For example, the introducednucleic acid molecule may encode a transcription factor that activatesor upregulates transcription of the protein of interest. Expression ofsuch a transcription factor in turn leads to expression, or more robustexpression of the protein of interest.

In certain embodiments, a nucleic acid that directs expression of theprotein is stably introduced into the host cell. In certain embodiments,a nucleic acid that directs expression of the protein is transientlyintroduced into the host cell. One of ordinary skill in the art will beable to choose whether to stably or transiently introduce a nucleic acidinto the cell based on his or her experimental needs. A gene encoding aprotein of interest may optionally be linked to one or more regulatorygenetic control elements. In certain embodiments, a genetic controlelement directs constitutive expression of the protein. In certainembodiments, a genetic control element that provides inducibleexpression of a gene encoding the protein of interest can be used. Theuse of an inducible genetic control element (e.g., an induciblepromoter) allows for modulation of the production of the protein in thecell. Non-limiting examples of potentially useful inducible geneticcontrol elements for use in eukaryotic cells include hormone-regulatedelements (e.g., see Mader, S. and White, J. H., Proc. Natl. Acad. Sci.USA 90:5603-5607, 1993), synthetic ligand-regulated elements (see, e.g.Spencer, D. M. et al., Science 262:1019-1024, 1993) and ionizingradiation-regulated elements (e.g., see Manome, Y. et al., Biochemistry32:10607-10613, 1993; Datta, R. et al., Proc. Natl. Acad. Sci. USA89:10149-10153, 1992). Additional cell-specific or other regulatorysystems known in the art may be used in accordance with the invention.

One of ordinary skill in the art will be able to choose and, optionally,to appropriately modify the method of introducing genes that cause thecell to express the protein of interest in accordance with the teachingsof the present invention.

Isolation of the Expressed Protein

In general, it will typically be desirable to isolate and/or purifyproteins expressed according to the present invention. In certainembodiments, the expressed protein is secreted into the medium and thuscells and other solids may be removed, as by centrifugation or filteringfor example, as a first step in the purification process.

Alternatively, the expressed protein may be bound to the surface of thehost cell. For example, the media may be removed and the host cellsexpressing the protein are lysed as a first step in the purificationprocess. Lysis of mammalian host cells can be achieved by any number ofmeans well known to those of ordinary skill in the art, includingphysical disruption by glass beads and exposure to high pH conditions.

The expressed protein may be isolated and purified by standard methodsincluding, but not limited to, chromatography (e.g., ion exchange,affinity, size exclusion, and hydroxyapatite chromatography), gelfiltration, centrifugation, or differential solubility, ethanolprecipitation and/or by any other available technique for thepurification of proteins (See, e.g., Scopes, Protein PurificationPrinciples and Practice 2nd Edition, Springer-Verlag, New York, 1987;Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A PracticalApproach, Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I.,Abelson, J. N. (eds.), Guide to Protein Purification: Methods inEnzymology (Methods in Enzymology Series, Vol. 182), Academic Press,1997, each of which is incorporated herein by reference). Forimmunoaffinity chromatography in particular, the protein may be isolatedby binding it to an affinity column comprising antibodies that wereraised against that protein and were affixed to a stationary support.Alternatively, affinity tags such as an influenza coat sequence,poly-histidine, or glutathione-S-transferase can be attached to theprotein by standard recombinant techniques to allow for easypurification by passage over the appropriate affinity column. Proteaseinhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin,pepstatin or aprotinin may be added at any or all stages in order toreduce or eliminate degradation of the protein during the purificationprocess. Protease inhibitors are particularly advantageous when cellsmust be lysed in order to isolate and purify the expressed protein.

One of ordinary skill in the art will appreciate that the exactpurification technique will vary depending on the character of theprotein to be purified, the character of the cells from which theprotein is expressed, and/or the composition of the medium in which thecells were grown.

Pharmaceutical Formulations

In certain preferred embodiments of the invention, produced polypeptidesor proteins will have pharmacologic activity and will be useful in thepreparation of pharmaceuticals. Inventive compositions as describedabove may be administered to a subject or may first be formulated fordelivery by any available route including, but not limited to parenteral(e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial,ophthalmic, transdermal (topical), transmucosal, rectal, and vaginalroutes. Inventive pharmaceutical compositions typically include apurified polypeptide or protein expressed from a mammalian cell line, adelivery agent (i.e., a cationic polymer, peptide molecular transporter,surfactant, etc., as described above) in combination with apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). In all cases, the composition should be sterile and should befluid to the extent that easy syringability exists. Preferredpharmaceutical formulations are stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. In general, therelevant carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thepurified polypeptide or protein in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the purified polypeptide or protein expressedfrom a mammalian cell line into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. Oral compositions generally includean inert diluent or an edible carrier. For the purpose of oraltherapeutic administration, the purified polypeptide or protein can beincorporated with excipients and used in the form of tablets, troches,or capsules, e.g., gelatin capsules. Oral compositions can also beprepared using a fluid carrier for use as a mouthwash. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches and thelike can contain any of the following ingredients, or compounds of asimilar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. Formulations for oral delivery may advantageouslyincorporate agents to improve stability within the gastrointestinaltract and/or to enhance absorption.

For administration by inhalation, the inventive compositions comprisinga purified polypeptide or protein expressed from a mammalian cell lineand a delivery agent are preferably delivered in the form of an aerosolspray from a pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer. Thepresent invention particularly contemplates delivery of the compositionsusing a nasal spray, inhaler, or other direct delivery to the upperand/or lower airway. Intranasal administration of DNA vaccines directedagainst influenza viruses has been shown to induce CD8 T cell responses,indicating that at least some cells in the respiratory tract can take upDNA when delivered by this route, and the delivery agents of theinvention will enhance cellular uptake. According to certain embodimentsof the invention the compositions comprising a purified polypeptideexpressed from a mammalian cell line and a delivery agent are formulatedas large porous particles for aerosol administration.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the purified polypeptide or protein anddelivery agents are formulated into ointments, salves, gels, or creamsas generally known in the art.

The compositions can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

In some embodiments, the compositions are prepared with carriers thatwill protect the polypeptide or protein against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active polypeptide or proteincalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier.

The polypeptide or protein expressed according to the present inventioncan be administered at various intervals and over different periods oftime as required, e.g., one time per week for between about 1 to 10weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or6 weeks, etc. The skilled artisan will appreciate that certain factorscan influence the dosage and timing required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Generally, treatment of a subjectwith a polypeptide or protein as described herein can include a singletreatment or, in many cases, can include a series of treatments. It isfurthermore understood that appropriate doses may depend upon thepotency of the polypeptide or protein and may optionally be tailored tothe particular recipient, for example, through administration ofincreasing doses until a preselected desired response is achieved. It isunderstood that the specific dose level for any particular animalsubject may depend upon a variety of factors including the activity ofthe specific polypeptide or protein employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

The present invention includes the use of compositions for treatment ofnonhuman animals. Accordingly, doses and methods of administration maybe selected in accordance with known principles of veterinarypharmacology and medicine. Guidance may be found, for example, in Adams,R. (ed.), Veterinary Pharmacology and Therapeutics, 8^(th) edition, IowaState University Press; ISBN: 0813817439; 2001.

Pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims. The contents of all figures and all references, patentsand published patent applications cited throughout this application areexpressly incorporated herein by reference.

EXAMPLES Example 1: Identification of the Metabolic Byproducts,Accumulating in Fed-Batch Cultures, which have Inhibitory Effects onGrowth of Mammalian Cells in Culture

Goal:

This experiment was carried out to identify the major growth inhibitors(metabolic byproducts) accumulating in the glucose restricted andconventional fed-batch cultures of mammalian cells, using globalmetabolite profiling approaches.

Materials and Methods:

Cells and Medium

CHO cells comprising a glutamine synthase expression system(commercially available from Lonza) (hereafter GS-CHO, Cell line A) andexpressing a recombinant antibody were used in the current experiment.Two types of medium were used in this experiment. First medium is“Medium A” which is used for inoculation of the experiment on day 0 ofthe culture. Second medium is “Medium B” which is the enriched nutrientmedia used as a feed medium for conventional and HIPDOG fed-batchprocesses for culture (described in the next section). Medium A is afortified version of insulin-free Medium 9 (U.S. Pat. No. 7,294,484,table 14), with slight differences in concentrations of sodiumbicarbonate and potassium chloride, and containing Pluronic F68 insteadof polyvinyl alcohol. It was fortified by adding 10% glutamine-freeMedium 5 (U.S. Pat. No. 7,294,484, table 7), and by further raising theconcentrations of eight amino acids (Glu, Tyr, Gly, Phe, Pro, Thr, Trpand Val). The concentrations of amino acids are listed in the Table 1below.

TABLE 1 Concentration of Amino Acids in Medium A Amino AcidsConcentration in Medium A (mM) alanine 0.4 arginine 5.3 asparagine•H2O21.1 aspartic acid 2.3 cysteine•HCl•H2O 0.4 cystine•2HCl 1.5 glutamicacid 0 monosodium glutamate 2.0 glutamine 0 glycine 3.6histidine•HCl•H2O 2.7 isoleucine 5.4 leucine 9.4 lysine•HCl 8.9methionine 3.1 phenylalanine 4.5 proline 9.1 serine 11.8 threonine 10.8tryptophan 2.3 tyrosine•2Na•2H2O 5.1 valine 10.3

Medium B has the same composition as Medium 5 (U.S. Pat. No. 7,294,484,table 7), but with higher levels of the amino acids (by a factor of2.5). The concentrations of amino acids in Medium B are shown in Table2.

TABLE 2 Concentration of Amino Acids in Medium B Amino AcidsConcnetration in Medium B (mM) alanine 6.0 arginine 32.9 asparagine•H₂O54.0 aspartic acid 15.0 cysteine•HCl•H₂O 0.0 cystine•2HCl 4.7 glutamicacid 6.0 monosodium glutamate 0.0 glutamine 0.0 glycine 6.0histidine•HCl•H₂O 10.5 isoleucine 27.0 leucine 38.9 lysine•HCl 30.0methionine 12.0 phenylalanine 15.0 proline 18.0 serine 45.2 threonine24.0 tryptophan 4.8 tyrosine•2Na•2H₂O 12.0 valine 24.0Bioreactor Setup

Two conditions were employed including conventional fed-batch processand a glucose restricted fed-batch process. In the glucose restrictedfed batch process (hereafter HIPDOG culture), glucose was limited byusing the HIPDOG technology (Gagnon et al, 2011). The pH dead-band usedwhile the HIPDOG control was operational was 7.025+/−0.025.

The conventional process was identical to the HIPDOG process/culturewith respect to inoculum cell density targeted (1E6 cells/mL), the mediaused, culture volume (14 the amount of feed added daily to the culture,and the process parameters including the temperature (36.5 C), pH(6.9-7.2) and the agitation rate (267 rpm). The two cultures onlydiffered in their glucose levels. In the conventional culture, glucosewas maintained at greater than 2 g/L between days 2 through 5, while inthe HIPDOG culture glucose was consumed by the cells naturally until theglucose level fell to a point at which the cells began to also consumelactic acid (observed by a slight rise in pH of the culture) and theHIPDOG technology/feeding strategy commenced. Post day 5 the glucoselevels in both the conditions were maintained at concentrations above 2g/L by feeding glucose as necessary and were treated similarly until day12. Viable cell density, lactate and ammonia concentration in the cellculture medium were measured on a daily basis for both the conditions.The base medium used is Medium A and the feed medium used was Medium B.

For metabolomic analysis, spent medium samples and the cell pelletsamples were collected and analyzed from duplicate reactors runs,performed for each condition. Time points considered for the analysisinclude days 0, 2, 3, 5, 7, 9 and 10. Metabolomic approach used employedNMR (groups 4 and 5 of Table 3), LC/MS and GC/MS (groups 1 to 3 of table3) techniques to assess the relative levels of metabolites at differenttime points of the culture. The details of the sample preparation andthe type of equipment/methods used for NMR, LC/MS and GC/MS analysis aredescribed below. The relative levels (fold changes) of all metaboliteswere measured and calculated. The relative levels were determined inboth the spent medium and cell pellet samples, which were calculatedbased on fold changes compared to the level of the metabolite when firstdetected. The fold changes were used to identify the metabolites thatwere accumulating to very high levels by day 7 of the HIPDOG and theconventional fed-batch culture.

Methods for Metabolomic Analysis

Liquid/Gas Chromatography with Mass Spectrometry

Sample preparation was conducted using a methanol extraction to removethe protein fraction while allowing maximum recovery of small molecules.The resulting extract was dried under vacuum and subsequently used forsample preparation for the appropriate instrument, either LC/MS orGC/MS.

The LC/MS portion of the platform was based on a Waters ACQUITY UPLC anda Thermo-Finnigan LTQ mass spectrometer, which consisted of anelectrospray ionization (ESI) source and linear ion-trap (LIT) massanalyzer. The sample was analyzed independently in both positive andnegative ion modes. Sample was reconstituted in acidic conditions forpositive ion mode and was gradient eluted using water and methanol, bothcontaining 0.1% Formic acid, whereas for negative ion mode sample wasreconstituted in basic extracts, which also used water/methanol,contained 6.5 mM ammonium bicarbonate for gradient elution. The MSanalysis alternated between MS and data-dependent MS² scans usingdynamic exclusion.

The samples destined for GC/MS analysis were re-dried under vacuumdesiccation for a minimum of 24 hours prior to being derivatized underdried nitrogen using bistrimethyl-silyl-triflouroacetamide (BSTFA). TheGC column was 5% phenyl and the temperature ramp is from 40° to 300° C.in a 16 minute period. Samples were analyzed on a Thermo-Finnigan TraceDSQ fast-scanning single-quadrupole mass spectrometer using electronimpact ionization. The instrument was tuned and calibrated for massresolution and mass accuracy on a frequent basis.

The data was extracted from the raw mass spec data files and peaks wereidentified. Subsequently, the peaks were annotated and quantified(arbitrary intensity values) with compound information by comparison tolibrary entries of purified standards or recurrent unknown entities. Thecombination of chromatographic properties and mass spectra gave anindication of a match to the specific compound or an isobaric entity.

NMR Sample Preparation, Data Acquisition and Processing

1000 μL of each sample was filtered using Nanosep 3K Omegamicrocentrifuge filter tubes for 60 minutes, and 630 μL of the filteredsample was used for NMR analysis. These filters are preserved withglycerol, and as such some trace amounts of glycerol may appear in theanalysis. Internal standard solution was added to each sample solution,and the resulting mixture was vortexed for 30 s. 700 μL of thecentrifuged solution was transferred to an NMR tube for dataacquisition.

NMR spectra were acquired on a Varian four-channel VNMRS 700 MHz NMRspectrometer equipped with a cryogenically cooled 1H/13C tripleresonance biomolecular probe with auto tuning. The pulse sequence usedwas a 1 D-tnnoesy with a 990 ms presaturation on water and a 4 sacquisition time. Spectra were collected with 32 transients and 4steady-state scans at 298 K.

Spectra were processed and .cnx files were generated using the Processormodule in Chenomx NMR Suite 8.0. Compounds were identified andquantified using the Profiler module in Chenomx NMR Suite 8.0 with theChenomx Compound Library version 9, containing 332 compounds. Forreporting purposes, the profiled concentrations have been corrected toreflect the composition of the original sample, instead of the contentsof the NMR tube. During sample preparation, each sample is diluted byintroducing an internal standard and, where necessary, to increase theanalyzed volume of a small sample.

Results:

Initially cells grew exponentially in both conventional and HIPDOGcultures and attained peak cells densities on day 6 and day 7,respectively, with HIPDOG culture peaking at much higher cell densities(FIG. 1 ). The lactate levels in the HIPDOG process/culture remained lowdue to application of the HIPDOG control (between day 2-day 5) whereasthe lactate levels accumulated to very high levels in case of theconventional fed-batch culture. Ammonia was also maintained at lowlevels during the conventional and HIPDOG culture by the use of cellscomprising a glutamine synthetase expression system. The titer (amountof protein of interest per liter of cell culture medium) was measured atthe end of the culture (Day 12). The HIPDOG culture attained highertiter compared to the conventional process. The differences in the celldensities and titer values are likely an outcome of the differences inthe lactate accumulations observed between the two cultures.

The metabolites that were accumulating to high levels on day 7 of theHIPDOG culture were identified based on the fold changes measured usingthe global metabolite profiling techniques. For each of the aboveidentified metabolites, the concentration at which the metaboliteaffects the growth of the cell negatively was determined throughspike-in experiments using purified compounds (see example 3). Theresults of these experiments were used to narrow the list of theputative novel inhibitors. A total of 9 inhibitors were identified bythis process. The list of the 9 metabolites identified as potentialinhibitors as well as their potential metabolic source in the cellculture medium are reported in Table 3.

Table 3 Shows the Names and the Functional Classes of the NineMetabolites Identified as Putative Growth Inhibitors Accumulating in theGS-CHO Fed-Batch Cultures

Functional Class/Metabolic Group Metabolite Source 13-(4-hydroxyphenyl)lactate Phenylalanine & tyrosine (HPLA) metabolism4-hydroxyphenylpyruvate Phenyllactate (PLA) Phenylalanine metabolism 2Indolelactate (indole-3-lactate) Tryptophan metabolism Indolecarboxylicacid (indole-3- carboxylic acid) 3 Homocysteine Methionine metabolism2-hydroxybutyric acid 4 Isovalerate Leucine 5 Formate Serine, Threonineand Glycine

Example 2: Determination of the Concentration to which the PutativeInhibitors Accumulate in Late Stages of HIPDOG Fed-Batch Cultures

Goal:

This experiment was carried out to assess the concentrations of newlyidentified putative growth inhibitors (metabolic byproducts) atdifferent time points in the HIPDOG fed-batch cultures of GS-CHO cells.

Materials and Methods:

Experimental setup is same as the one defined in Example 1.Quantification was performed by LC/MS and GC/MS methods for metabolitesin the first two groups of Table 3 and NMR technology was used forquantification of the metabolites in groups 4 and 5. For first twogroups of the metabolites listed in the metabolite column of Table 3,purified compounds were obtained commercially and solutions of thesecompounds at known concentrations were prepared using the Medium A asthe base solvent. Using a similar LC/MS and GC/MS global metaboliteprofiling approach as that used for inhibitor identification andrelative quantification (see Example 1), independent calibration curvesfor four metabolites were prepared. These calibration curves aremathematical correlations of the actual amounts of the metabolite usedin LC/MS and GS/MS techniques to the intensity values generated by thesame. The correlations are subsequently used along with the intensityvalues generated in Example 1 to calculate the concentration of themetabolites at different time points in the culture.

Results:

The concentrations of newly identified metabolites from the first twogroups of Table 3 were determined using the calibration curvesdeveloped, and the concentrations for the metabolites listed in groups 4and 5 were determined by NMR technology as discussed in the Materialsand Methods section of Example 1. The concentration of six putativeinhibitors (phenyllactate, 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate, indolelactate, isovalerate and formate) on day7 of the HIPDOG fed-batch cultures are listed Table 4.

TABLE 4 concentrations of six metabolites on day 7 of the HIPDOG cultureDay 7 Concentration Metabolite (mM) 3-(4hydroxyphenyl)lactate (HPLA)0.38 4-hydroxyphenylpyruvate 0.08 Phenyl lactate (PLA) 0.20Indolelactate 0.26 Isovalerate 2.41 Formate 3.97

Example 3: Experiment to Establish the Growth Suppressive Effect ofIdentified Putative Inhibitors at the Concentrations Detected on Day 7of the HIPDOG Fed-Batch Culture

Goal:

This experiment was carried out to assess the effect of the newlyidentified putative inhibitors, at the concentrations determined on day7 of the HIPDOG culture, on growth of GS-CHO cells in culture. Theindependent effect of the nine metabolites (phenyllactate,3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, indolelactate,indolecarboxylic acid, homocysteine, 2-hydroxybutyric acid, isovalerateand formate) on growth of cells was tested first. Subsequently,synergistic effect for four metabolites (phenyllactate,3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate and indolelactate)on cell growth was tested.

Materials and Methods:

GS-CHO cells producing a recombinant antibody were inoculated at lowviable cell densities (0.1E6 cells/mL) in various conditions in 5 mlvolume, 6-well plate cultures. These conditions include fresh Medium Aor fresh Medium A spiked-in with four putative inhibitors atconcentrations detected on day 7 of the HIPDOG culture of Example 1(phenyllactate at 0.2 mM, 3-(4-hydroxyphenyl)lactate at 0.38 mM,4-hydroxyphenylpyruvate at 0.08 mM and indolelactate at 0.26 mM). In aseparate experiment, GS-CHO cells producing a recombinant antibody wereinoculated at low viable cell densities in fresh Medium A spiked-in withdifferent concentrations of 9 inhibitors (indolecarboxylic acid,homocysteine, 2-hydroxybutyric acid, phenyllactate,3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, indolelactate,isovalerate and formate) with one inhibitor at a time per condition. ThepH of the Medium A, spiked-in with any one of the nine metabolites orany combination of the nine metabolites, was adjusted to 7 beforeinoculating the cells (on day 0). The concentrations tested are:

-   -   indolecarboxylic acid: 0, 0.5 and 1 mM    -   homocysteine: 0, 0.5 and 1 mM    -   2-hydroxybutyric acid: 0, 1 and 5 mM    -   phenyllactate: 0, 1 and 5 mM    -   3-(4-hydroxyphenyl)lactate: 0, 0.1, 0.3, 0.5, 1 and 5 mM    -   4-hydroxyphenylpyruvate: 0, 0.05, 0.1 and 0.25 mM    -   indolelactate: 0, 1 and 3 mM    -   isovalerate: 0, 1, 2.5 and 5 mM    -   formate: 0, 2, 4 and 6 mM

For indolelactate, the stock solution (500 mM) was prepared in DMSO.Hence, pure DMSO spike-in conditions (‘DMSO cont’ or DMSO control) wereincluded for every concentration of indolelactate tested, so as tocontrol for the effect of DMSO on the growth of the cells. All theconditions were run in duplicates or triplicates. Growth of the cells inabove described conditions was monitored for 5 or 6 days.

Results:

The independent effect of the all the nine inhibitors on growth of theGS-CHO cells was investigated (FIG. 2 , FIG. 3 , FIG. 4 and FIG. 5 ).

Indolecarboxylic acid and 4-hydroxyphenylpyruvate were observed to havea potent negative effect on growth of cells at concentrations of 1 mM orlower. Modest inhibition of growth was observed when GS-CHO cells wereexposed to either homocysteine, 2-hydroxybutyric acid or3-(4-hydroxyphenyl)lactate at concentrations higher than 0.5 mM or 1 mM.L-phenyllactate had a mild effect on growth of cells at 1 mMconcentration. Indolelactate showed little or no effect on growth ofcells at concentrations of 3 mM or below. Formate had a negative effecton growth at concentrations 2 mM and beyond. Isovalerate had asignificant negative effect on growth of cells at concentrations above 1mM.

Earlier experiments showed that the concentration at which cell growthis inhibited was much higher when each of the nine putative inhibitorswere added independently than the concentration observed on day 7 of theHIPDOG fed-batch culture. Therefore, the effect of these inhibitors whentreated in combination was subsequently investigated. Interestingly, ontreating the cells with the combination of four [phenyllactate,4-hydroxyphenylpyruvate, 3-(4-hydroxyphenyl)lactate, indolelactate] ofthe nine metabolites, at concentrations detected on day 7 of HIPDOGculture, the cell growth was significantly inhibited when compared tocell growth in the fresh medium (FIG. 6 ). This data indicates that theabove four metabolites act in a synergistic manner to inhibit the growthof the cells. The above mentioned nine metabolites are by-products ofphenylalanine, tyrosine, tryptophan, leucine, serine, threoninemethionine and glycine metabolism. Identifying the enzymes in thesepathways that contribute to the biosynthesis of the inhibitors providesa strategy to genetically modify the cells to suppress the production ofthe same by gene modification.

Since these four metabolites are by-products of phenylalanine, tyrosineand tryptophan metabolism, reducing the concentrations of theseprecursor amino acids in culture can limit the formation ofcorresponding inhibitory metabolites. Further, since methioninemetabolites (homocysteine and 2-hydroxybutyrate) and leucine, serine,threonine and glycine metabolites (isovalerate and formate) also have anegative effect on cell growth, reducing methionine, leucine, serine,threonine and glycine levels in culture could potentially limit theformation of these metabolites and promote cell growth.

Example 4: Reduction of the Growth Suppressive Effect of the NewlyIdentified Inhibitors by Nutrient Limitation Strategies in Fed-BatchCulture

Goal:

This experiment was performed to reduce the formation of newlyidentified inhibitors by limiting the supply of the carbon sourcesresponsible for their biosynthesis. The goal of this experiment was toassess if such reduction in inhibitor formation relieves the growthsuppression in the late stages of the culture, resulting in increasedmaximum viable cell densities in the fed-batch cultures.

Materials and Methods:

Cells and Bioreactor Setup

GS-CHO cells expressing a recombinant antibody (cell line A) were usedin the current experiment. Two conditions were tested as part of thisexperiment: A) HIPDOG fed-batch culture with low levels of four aminoacids ((tyrosine, methionine, phenylalanine and tryptophan) (Low AAcondition)), B) HIPDOG fed-batch culture with normal amino acidsconcentrations (control HIPDOG condition/culture). Exponentially growingcells from seed culture were inoculated at 1×10⁶ cells/mL into eachproduction bioreactor. For both the conditions, HIPDOG strategy was inoperation between day 2 and day 7 of the culture. In the low amino acidcondition, the concentrations of tyrosine, tryptophan, phenylalanine andmethionine were maintained between 0.5 mM and 1 mM for first seven daysof the culture after which they were adjusted to the levels of each ofthose amino acids in the control HIPDOG condition. Post day 7 both theconditions were treated similarly. Viable cell density, lactate, ammoniaand amino acid concentrations were measured on a daily basis (aminoacids measured only for first seven days). For both conditions, theinoculum viable cell density targeted (1×10⁶ cells/mL), and the culturevolume (1 L) and the process parameters including the temperature (36.5C), pH (6.9-7.2) and the agitation rate (267 rpm) were identical. Thebase medium used in the control HIPDOG condition was Medium A and thatused in Low AA condition was the modified version of Medium A with lowconcentrations of the four amino acids (tyrosine, tryptophan,phenylalanine and methionine at approximately 0.6 mM). The feed mediumused for control HIPDOG culture was Medium B. For the Low AA condition,either the original Medium B or a modified version of Medium B withhigher concentration of four amino acids (tyrosine, tryptophan,methionine and phenylalanine) was formulated and used as feed media (60%higher for methionine and ˜100% higher levels for tyrosine, tryptophanand phenylalanine as compared to original feed Medium B). The levels ofthe four amino acids in the modified version of Medium B was configuredbased on the cell specific consumption rates for the four amino acidsand the previously determined feeding schedule for the cell line A in aHIPDOG process of culture. Amino acid concentrations were measured everyday using a UPLC based amino acid method which is described in detailbelow. Based on the level of amino acids at a given sampling point andthe feeding schedule, one of the two types of medium B (original orhigher concentration) was chosen as the feed medium till next samplingpoint such that the concentration for the four amino acids are between0.5 mM-1 mM at the next sampling time point.

Amino Acid Analysis

10 μL of either a standard amino acid mix solution or a spent mediumsample (10 times diluted sample) was mixed with 70 μL of AccQ●Tag Ultraborate buffer (Waters UPLC AAA H-Class Applications Kit [176002983]),and 20 μL of AccQ●Tag reagent previously dissolved in 1.0 mL of AccQ●TagUltra reagent diluent was added. The reaction was allowed to proceed for10 min at 55° C. Liquid chromatographic analysis was performed on aWaters Acquity UPLC system, equipped with a binary solvent manager, anautosampler, a column heater and a PDA detector. The separation columnwas a Waters AccQ●Tag Ultra column (2.1 mm i.d.×100 mm, 1.7 μmparticles). The column heater was set at 55° C., and the mobile phaseflow rate was maintained at 0.7 mL/min. Eluent A was 10% AccQ●Tag Ultraconcentrate solvent A, and eluent B was 100% AccQ●Tag Ultra solvent B.The nonlinear separation gradient was 0-0.54 min (99.9% A), 5.74 min(90.0% A), 7.74 min (78.8% A), 8.04-8.64 min (40.4% A), 8.73-10 min(99.9% A). One microliter of sample was injected for analysis. The PDAdetector was set at 260 nm. The previously determined elution times forthe amino acids are used to identify the specific amino acid peaks onthe chromatogram for each sample. The amino acid concentrations wereestimated using the area under the peak and the calibration curvegenerated using the standard solution (Amino Acids Standard H, ThermoScientific, PI-20088).

Results:

The concentrations of the amino acids were successfully maintainedbetween 0.5 mM-1 mM in Low AA conditions (FIG. 8 and FIG. 9 ). The aminoacid concentrations in the control HIPDOG process remained high over thecourse of the culture. As shown in FIG. 7 , the cell densities in theLow AA condition peaked around 35×10⁶ cells/ml on day 7 whereas the celldensities in control HIPDOG condition peaked around 32×10⁶ cells/mL. Theharvest titer levels in the Low AA condition (5.3 g/L) were 18% higherthan the control condition (4.5 g/L). Clearly, limiting the amino acidsupply increased the cell densities (and thereby the titer) in the latestages of the cultures.

Example 5 demonstrates that such an increase in growth and productivitycan be explained as a result of reduced production of the newlyidentified inhibitors.

Example 5: Demonstrating (i) the Reduced Accumulation of the NewlyIdentified Inhibitors Through the Limitation of Amino Acids and (ii) theReproducibility of the Positive Effect of Such Limitation of InhibitoryMetabolites on Growth of GS-CHO Cells (Cell Line A) in Fed-BatchCultures

Goal:

The main goal of this example was to demonstrate the reduction in theaccumulation of the newly identified inhibitors in fed-batch cultures bylimiting the supply of the carbon sources (amino acids) responsible fortheir biosynthesis. In this example, two experiments are included todemonstrate that such reduction in the levels of the newly identifiedinhibitors through limitation of four amino acids or eight amino acidsrelieves the growth suppression in the late stages of the fed-batchculture resulting in increased maximum viable cell densities.

Materials and Methods:

Cells and Bioreactor Setup Cell line A was used in the two experimentsperformed as part of this example.

Three conditions were tested in the first experiment:

A) HIPDOG fed-batch culture with low levels of tyrosine, methionine,phenylalanine and tryptophan (Low 4AA+HIPDOG condition for culture),

B) HIPDOG fed-batch culture with low levels of tyrosine, methionine,phenylalanine, tryptophan, leucine, serine, threonine and glycine (Low8AA+HIPDOG condition), and,

C) two HIPDOG fed-batch cultures with normal amino acids concentrations(HIPDOG 1 and HIPDOG 2).

In a second experiment two conditions were tested:

A) HIPDOG fed-batch culture with low levels of tyrosine, methionine,phenylalanine, tryptophan, leucine, serine, threonine and glycine (Low8AA+HIPDOG condition), and,

B) HIPDOG fed-batch culture with low levels of tyrosine, methionine,phenylalanine and tryptophan (Low 4AA+HIPDOG condition).

First experiment was run for 12 days whereas the second experiment wasrun for 16 days.

In both the experiments, exponentially growing cells from seed cultureswere inoculated at 1×10⁶ cells/mL into each production bioreactor. Forall the conditions, HIPDOG control was in operation between day 2 andday 7 of the culture. In the low amino acid conditions, theconcentrations of above mentioned four or eight amino acids weremaintained between 0.5 mM and 1 mM for first seven days of the cultureafter which they were adjusted to the levels closer to the respectiveamino acids in the control HIPDOG conditions. Post day 7, both theconditions were treated similarly. Viable cell, ammonia and amino acidconcentrations were measured on daily basis. For all the conditions, theinoculum viable cell density targeted (1×10⁶ cells/mL), the culturevolume (1 L), and the process parameters including the temperature (36.5C), pH (6.9-7.2) and agitation rate (259 rpm) were identical. The basemedium used in the HIPDOG condition was Medium A and that used in lowamino acid conditions was the modified version of Medium A with lowconcentrations of either four amino acids (tyrosine, tryptophan,phenylalanine and methionine at approximately 0.6 mM) or eight aminoacids (tyrosine, tryptophan, phenylalanine, methionine, leucine, serine,threonine and glycine at approximately 0.6 mM). The feed medium used forall the conditions was Medium B. Amino acid concentrations were measuredevery day using UPLC based amino acid method as described in Example 4.In the low amino acid conditions, based on the level of amino acids at agiven sampling point and the feeding schedule to be followed,concentrated solutions of the amino acids were supplemented to theconditions such that the concentration for the four amino acids or theeight amino acids in corresponding low amino acid conditions are between0.5 mM-1 mM at the next sampling time point. Spent medium samples fromvarious conditions across both the experiments were analyzed for thelevels of the newly identified inhibitors using the NMR technologydescribed in the Materials and Methods section of Example 1.

Results:

In the first experiment, the concentrations of the amino acids weresuccessfully maintained between 0.5 mM-1 mM in both Low 4 AA and Low 8AA conditions until day 7 of the fed-batch cultures (FIGS. 11-14 ). Suchlimitation of amino acid levels in the two conditions resulted in lowerlevels of accumulation of the newly identified metabolites (FIG. 15 andFIG. 16 ). Isovalerate, formate, 3-(4-hydroxyphenly)lactate andindole-3-lactate were specifically profiled. Isovalerate and formate arebyproducts of leucine, serine, glycine and threonine, which arecontrolled at low levels only in Low 8 AA condition. These amino acidsare not controlled at low levels in the Low 4 AA condition.Correspondingly, significantly lower concentrations of isovalerate wereonly seen in the Low 8 AA condition. The levels isovalerate were higherin control HIPDOG conditions and the Low 4 AA condition (FIG. 15B).Formate levels were similar across the all conditions on day 7 of theculture; however, on a per cell basis the amount of formate produced islower in Low 8 AA condition compared to HIPDOG conditions. The other twoinhibitors profiled, 3-(4-hydroxyphenly)lactate and indole-3-lactate,are byproducts of the amino acids phenylalanine and tryptophan, whichare controlled at lower levels in both Low 8 AA and Low 4 AA conditions.Significantly lower concentrations of these two inhibitors were observedin both Low 8 AA and Low 4 AA conditions compared the HIDPOG conditionsat day 7 (FIGS. 15A and 16 ). The cells in Low 8 AA and Low 4 AAconditions grew better than the control conditions (HIPDOG1 and HIPDOG2) peaking at cell densities 50×10⁶ cells/mL and 45×10⁶ cells/mL,respectively, on day 9 whereas the cell densities in control HIPDOGconditions peaked around 32×10⁶ cells/mL (FIG. 10A). Such an increase inthe cell growth observed in the late stages of the low amino acidconditions can be explained as an outcome of the reduced inhibitoraccumulations in the culture (FIGS. 15 and 16 ). In addition, the lowamino conditions had higher titer compared to the control HIPDOGconditions until day 9, which then tapered off to match the titer levelsof HIPDOG conditions by the end of the culture (FIG. 10B). The post day9 reduction in the protein production in the low amino acid conditionswas attributed to the near exhaustion of tyrosine levels in the culturespost day 9 (FIG. 11A).

The second experiment was performed to verify and reproduce theincreased positive effect of limiting leucine, serine, glycine andthreonine in Low 8 AA condition when compared to Low 4 AA condition(FIG. 17 ). Only two conditions were tested in this experiment includingthe Low 8 AA and Low 4 AA condition using the cell line A. The eightamino acids or the four amino acids were controlled between 0.5 mM and 1mM until day 7 of the culture. Amino acid data is not shown for thisexperiment but is similar to the amino acid profiles observed for thetwo conditions in above experiment (FIGS. 11-14 ) except for tyrosinewhich was not exhausted in this experiment. The cells grew better in theLow 8 AA condition peaking at 42×10⁶ cells/mL on day 9 whereas the celldensities in Low 4 AA condition peaked around 33×10⁶ cells/mL (FIG. 17). Such an increased cell density also translated into higher levels ofharvest titer in the Low 8 AA conditions when compared to Low 4 AAcondition.

Example 6: Demonstrating (i) the Reduction in the Accumulation of theNewly Identified Inhibitors Through the Limitation of Amino Acids and(ii) the Positive Effect of Such Limitation of Inhibitory Metabolites onGrowth of a Different GS-CHO Cell Line (Cell Line B) in Fed-BatchCultures

Goal:

This experiment was performed to demonstrate that the growth beneficialeffects of limiting the levels of certain amino acids on the growth ofcells were not specific to one cell line (cell line A) but are moregeneral and can be applied to other cell lines. Two nutrient limitationsexperiments were performed as part of this example using a different CHOcell line (cell line B) producing a different recombinant antibody. Thegoal of these experiments was to show that in fed-batch cultures, thecontrol of amino acids at lower levels results in reduced inhibitoraccumulations and such low accumulations of inhibitors can explain theincreased viable cell densities and protein titers that were seen in thelow amino acid fed-batch cultures.

Materials and Methods:

Cells and Bioreactor Setup

A new GS-CHO cell line (cell line B) expressing a different recombinantantibody was used in this example. Two experiments were performed tounderstand the effect of simultaneously limiting either four or eightamino acids. In first experiment, two conditions were tested: A) HIPDOGfed-batch culture with low levels of eight amino acids includingtyrosine, methionine, phenylalanine, tryptophan, leucine, serine,glycine and threonine (Low 8 AA+HIPDOG condition) and B) HIPDOGfed-batch culture with normal amino acids concentrations (HIPDOGcondition). In the second experiment, two conditions were tested: A)HIPDOG fed-batch culture with low levels of four amino acids includingtyrosine, methionine, phenylalanine and tryptophan (Low 4 AA+HIPDOGcondition) and B) HIPDOG fed-batch culture with normal amino acidsconcentrations (HIPDOG condition). First experiment was run for 12 dayswhereas the second experiment was only run till day 8 of the culture.

Exponentially growing cells from seed cultures were inoculated at 1×10⁶cells/mL into each production bioreactor. For all the conditions, HIPDOGstrategy was in operation between day 2 and day 7 of the culture. In thelow amino acid conditions, the concentrations of above mentioned four oreight amino acids were maintained between 0.5 mM and 1 mM for firstseven days of the culture after which they were adjusted to the levelscloser to the respective amino acids in the control HIPDOG condition.Post day 7, both the conditions were treated similarly. Viable cell,ammonia and amino acid concentrations were measured on daily basisthroughout the culture. For all the conditions, the inoculum viable celldensity targeted (1×10⁶ cells/mL), and the culture volume (1 L) and theprocess parameters including the temperature (36.5 C), pH (6.9-7.2) andthe agitation rate (259 rpm) were identical. The base medium used in theHIPDOG condition was Medium A and that used in Low AA conditions was themodified version of Medium A with low concentrations of either fouramino acids (tyrosine, tryptophan, phenylalanine and methionine atapproximately 0.6 mM) or eight amino acids (tyrosine, tryptophan,phenylalanine, methionine, leucine, serine, threonine and glycine atapproximately 0.6 mM). The feed medium used for all the conditions wasMedium B. Amino acid concentrations were measured every day using UPLCbased amino acid method as described in Example 4. In the low amino acidconditions, based on the level of amino acids at a given sampling pointand the feeding schedule to be followed, concentrated solutions of theamino acids were supplemented to the conditions such that theconcentration for the four amino acids or the eight amino acids incorresponding conditions are between 0.5 mM-1 mM at the next samplingtime point. Spent medium samples from various conditions across both theexperiments of this Example were analyzed to quantitate the levels ofthe newly identified inhibitors using the NMR technology, as describedin the Materials and Methods section of Example 1.

Results:

In the first experiment, the concentrations of the eight amino acidswere successfully maintained between 0.5 mM-1 mM in Low 8 AA conditionuntil day 7 of the fed-batch cultures. Amino acid culture profiles arenot shown for this experiment but are similar to those observed for thesimilar conditions in Experiment 1 of Example 5 (FIGS. 11-14 ) exceptfor tyrosine which was not exhausted in this experiment. Such limitationof amino acid levels in the Low 8 AA condition resulted in lower levelsof biosynthesis and reduced accumulation of the newly identifiedmetabolites. Four of the nine inhibitors listed in Table 3 were profiled(FIG. 19 and FIG. 20 ). In Low 8 AA condition, significantly loweraccumulations of isovalerate, 3-(4-hydroxyphenly)lactate andindole-3-lactate were observed compared to control HIPDOG condition.Formate levels were observed to be higher in Low 8 AA condition on day10 of the culture, however, on a per cell basis, the amount of formateproduced was similar in the Low 8 AA condition compared to the HIPDOGcondition. The cells in Low 8 AA conditions grew better than the controlcondition (HIPDOG) peaking at cell densities of 40×10⁶ cells/mL on day 9whereas the cell densities in control HiPDOG conditions peaked around32×10⁶ cells/mL (FIG. 18A). Further, the increased growth observed inLow 8 AA condition translated into higher titer levels compared to thecontrol HIPDOG condition (FIG. 18B). Such an increase in the cell growthand productivity observed in the low amino acid condition can beexplained as an outcome of the reduced inhibitor biosynthesis andaccumulation (FIGS. 19 and 20 ).

The second experiment was performed to investigate the effect ofcontrolling the levels of four amino acids (tyrosine, phenylalanine,tryptophan and methionine) between 0.5 mM-1 mM (Low 4 AA+HIPDOG) ongrowth and productivity of cell line B, when compared to control HIPDOGcondition (FIGS. 21-23 ). Amino acid culture profiles are not shown forthis experiment but are similar to those observed for the similarconditions in Experiment 1 of Example 5 (FIGS. 11-14 ) except fortyrosine which was not exhausted in this experiment. Such limitation ofamino acid levels in the Low 4 AA condition resulted in lower levels ofbiosynthesis and reduced accumulation of the newly identifiedmetabolites. Four of the nine inhibitors listed in Table 3 were profiled(FIG. 22 and FIG. 23 ). Except isovalerate, the levels of the otherthree inhibitors were lower in the Low 4 AA condition compared to HIPDOGcondition. Leucine not being one of the amino acids which is controlledat low levels in the Low 4 AA condition, its metabolic intermediateisovalerate accumulates in Low 4 AA condition to levels similar to thoseseen the control HIPDOG condition. The cells in Low 4 AA conditions grewbetter peaking at cell densities 37×10⁶ cells/mL on day 9 of the culturewhereas the cell densities in control HIPDOG condition peaked around32×10⁶ cells/mL (FIG. 21A). Further, the increased growth observed inLow 4 AA condition translated into higher titer levels compared to thecontrol HIPDOG condition (FIG. 21B). Such an increase in the cell growthand productivity observed in the low amino acid condition can beexplained as an outcome of the reduced inhibitor biosynthesis andaccumulation (FIGS. 22 and 23 ).

Example 7: Use of RAMAN Spectroscopy for Online Measurement of the Four(Phenylalanine, Tyrosine, Tryptophan and Methionine) or Eight AminoAcids (Phenylalanine, Tyrosine, Tryptophan, Methionine, Leucine, Serine,Glycine, and Threonine) and Newly Identified Inhibitors

Raman spectroscopy is based on the inelastic scattering of monochromaticlight (photons) by a molecule. The technology uses the frequency shiftin the light, due to a change in energy of the photon when it isabsorbed and reemitted by the molecule, to determine the characteristicsof the molecule. This technology has been successfully used in microbialand mammalian cell culture bioreactors with success to measure thelevels of various process parameters. Raman spectroscopy has also beenused to determine the concentrations of glucose, lactate, ammonia,glutamine and glutamate in CHO cell cultures.

The Raman spectra for all the amino acids have been previously reportedin the literature. Raman spectra for the newly identified inhibitorymetabolites is characterized using a solution of the purifiedmetabolites. An empirical model is built by employing a training set ofspectral data generated using known concentrations of each of the fouror eight amino acids or the newly identified inhibitors, individually.The sample matrix (background) used for of the preparation ofcalibration samples (used as the training set) is a mix of spent mediasamples taken from different time points of different cell cultureprocesses. The model developed using such a training set is more generaland can be applied to any other cell culture process. This model is usedto measure the concentration of the four or eight amino acids or themetabolites (inhibitors) in the culture (online) and accordingly controlthe levels of amino acids through feedback control feeding strategies.

Example 8: Suppression of Inhibitor Formation Through Control of AminoAcids at Low Levels in Fed-Batch Cultures by Using Online Measurement ofInhibitor Concentration

A fed-batch process is designed to reduce the formation of theinhibitors through control of four (phenylalanine, tyrosine, tryptophanand methionine) or eight amino acids (phenylalanine, tyrosine,tryptophan, methionine, leucine, serine, glycine, and threonine) at lowlevels, for example between 0.2-1 mM in the cell culture medium. Such acontrol of the inhibitor production is attained by a feeding strategythat operates as a feed-back loop based on the online measurement of theinhibitors themselves. Such online measurements are in the form of RAMANspectroscopy or through use of HPLC/UPLC based technology with anauto-sampler that draws sample from reactor and transfers it to theequipment in a programmed manner. As and when the concentration of theinhibitors rises above a specified level (example: 0.2 mM), the amountof phenylalanine, tyrosine, tryptophan, methionine, leucine, serine,glycine, and threonine fed to the cells is decreased until theconcentration of inhibitors falls below a predefined level (for example0.1 mM).

Example 9: Suppression of Inhibitor Formation Through Online or OfflineMeasurement and Control of Amino Acids at Low Levels in Fed-BatchCultures

A fed-batch process is designed to reduce the formation of theinhibitors through control of the four (phenylalanine, tyrosine,tryptophan and methionine) or eight amino acids (phenylalanine,tyrosine, tryptophan, methionine, leucine, serine, glycine, andthreonine) at low levels (0.2-1 mM). Such a control of inhibitorformation is attained by a feeding strategy that operates as a feed-backloop based on the online measurement of the amino acids. Such onlinemeasurements is in the form of RAMAN spectroscopy or through use ofHPLC/UPLC based technology with an auto-sampler that draws sample fromreactor and transfers it to the equipment in a programmed manner. As andwhen the concentration of the amino acids falls below a specified level(example: 0.5 mM), estimated amounts of feed medium (similar to MediumB) is added to the culture so as to maintain the concentrations of thefour or eight amino acids.

Alternatively, the samples are taken on a once per day basis and aminoacid concentrations are measured offline using a UPLC/HPLC method. Theamino acid concentrations obtained are used to calculate the cellspecific uptake rates of the four (phenylalanine, tyrosine, tryptophanand methionine) or eight amino acids (phenylalanine, tyrosine,tryptophan, methionine, leucine, serine, glycine, and threonine) betweenprevious two sampling time points. Assuming that the cells maintain thesame specific rate of amino acid consumption until the next samplingtime point, the amount of feed medium (example: Medium B) to be addedtill the next sampling point is determined and provided to the cells,such that the concentrations of the amino acids are always within thedesired range (0.2 mM-1 mM).

Example 10: Suppression of Inhibitor Formation Through ProgrammedFeeding so as to Keep the Amino Acids at Low Levels in Fed-BatchCultures

The formation of the newly identified inhibitors in culture is kept lowby maintaining the concentration of the four (phenylalanine, tyrosine,tryptophan and methionine) or eight amino acids (phenylalanine,tyrosine, tryptophan, methionine, leucine, serine, glycine, andthreonine) at low levels in culture (0.2-1 mM). This is attained bydesigning a programmed feeding strategy (using modified Medium B) suchthat the concentrations of the amino acids, at any given time point inthe culture are always within the desired range (0.2 mM-1 mM). Such afeeding strategy is contrived by assessing the specific consumptionrates of amino acids at different time points along the culture andmodifying the concentration in the feed medium (Medium B) such that withthe above defined feeding strategy/schedule, sufficient amounts of aminoacids are provided to the culture to maintain the amino acidsconcentrations within the desired range (0.2-1 mM).

Example 11: Experiment to Determine the Gene Targets for MetabolicEngineering for Suppressing the Biosynthesis of the Inhibitors Relatedto Phenylalanine/Tyrosine Pathway

Goal:

The experiment was performed to determine the cause for production ofinhibitor molecules including phenyllactate, 4-hydroxyphenylpyruvate and3-(4-hydroxyphenyl)lactate. Gene expression of all the enzymes in thephenylalanine/tyrosine pathway was probed using Real Time QuantitativePolymerase Chain Reaction (RT-qPCR) assay. Based on the gene expressionand the biochemistry of the phenylalanine/tyrosine catabolic pathway,gene targets for metabolic engineering of the CHO cell line A thatsuppress the biosynthesis of above mentioned inhibitors, was identified.

Materials and Methods:

Gene Expression Analysis

RT-qPCR assay was used to assess relative gene expression levels ofenzymes in the leucine and phenylalanine/tyrosine metabolic pathways. RTqPCR measures transcript abundance, and hence, gene expression byamplifying a target cDNA sequence using PCR in combination with adetection reagent (i.e. SYBR Green). SYBR green is a molecule thatfluoresces when bound to double stranded DNA and the fluorescence can bemeasured in real time during the RT qPCR assay. The amount offluorescence is directly proportional to the amount of double strandedPCR product (also called amplicon) in the reaction. Relative geneexpression levels are determined by measuring the number of PCR cyclesrequired for SYBR green fluorescence to surpass the backgroundfluorescence and increase logarithmically. This cycle number is commonlyreferred to as the C_(T) (Threshold Cycle). A transcript in highabundance would have a lower C_(T) value as it would require fewer PCRcycles for the fluorescence to surpass the background fluorescencewhere, conversely, a transcript in lower abundance would have a higherC_(T) value as it would require more PCR cycles for the fluorescence tosurpass the background level.

The RT qPCR assay was performed using an Applied Biosystems 7500 RealTime PCR system (Applied Biosystems) and the PowerUP SYBR Green MasterMix reagent (Life Technologies). The PCR primers were designed using thePrimer3 algorithm based on genomic DNA sequences contained in theChinese Hamster Ovary (CHO) genome browser. RNA was prepared from CHOcell line A using the Qiagen RNeasy Kit (Qiagen) which was in turn usedas template for oligo dT primed cDNA synthesis using the SuperScript IIIFirst-Strand Synthesis System for RT-PCR (Life Technologies). The C_(T)values of the targeted metabolic genes were tabulated and compared tothe C_(T) value of a well characterized housekeeping gene, Beta Actin.The difference between the C_(T) of the target gene and the C_(T) ofBeta Actin was reported as the ΔC_(T). High ΔC_(T) value indicates lowgene expression level.

Results:

The C_(T) and ΔC_(T) values for the genes in the phenylalanine/tyrosinepathway is shown in FIG. 24 . The gene expression data from thephenylalanine/tyrosine pathway indicates that CHO cell line A has lowexpression of the PAH, HPD and HGD genes. As the expressed protein (orenzyme) levels correlate to the transcript levels, low levels of PAHenzyme can result in the phenylalanine being converted to phenylpyruvateand subsequently to phenyllactate by catalytic action of GOT1/GOT2 andMIF enzymes, both of which are expressed at high levels in CHO cell lineA. Similarly, due to low levels of HPD and HGD enzymes in CHO cell line,tyrosine is channeled towards production of 4-hydroxyphenylpyruvate and3-(4-hydroxyphenyl)lactate by catalytic action of GOT1/GOT2 and MIFenzymes. Reducing the channeling of the phenylalanine and tyrosine fluxtowards inhibitor production requires the down-regulation of theexpression of GOT1/GOT2 and MIF genes. However, GOT1/GOT2 and MIF arecritical for other physiologically important metabolic functions.Therefore, down-regulation of the two enzymes wouldn't potentially yielda viable cell line. An alternative way to reduce inhibitor biosynthesisis through overexpression of the PAH, HPD and HGD genes which would thenchannel the flux away from inhibitor production and towards productionof Krebs cycle metabolites (which are used for energy synthesis inmitochondria). Hence the metabolic targets for phenylalanine/tyrosinepathway are PAH, HPD and HGD genes.

Example 12: Experiment to Determine the Gene Targets for MetabolicEngineering for Suppressing the Biosynthesis of the Inhibitors Relatedto Leucine Pathway

Goal:

The experiment was performed to determine the cause for the biosynthesisand accumulation of isovaleric acid, which is a byproduct of leucinemetabolism and accumulates to very high levels in fed-batch cultures ofCHO cells. Gene expression of all the enzymes in the leucine pathway wasprobed using Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)assay. Further, production of isovaleric acid in human diseaseconditions such as isovaleric academia has been reported to be due to‘loss of function’ mutations in enzymes down stream of isovaryl-CoA, anintermediate in leucine catabolism pathway. These enzymes include Ivd,Mccc1, and Mccc2, which can harbor loss of activity mutations thoughexpressed at high levels. Therefore, the mutation status of theseenzymes in CHO cell line A was investigated. Based on the geneexpression analysis and mutation analysis of the above mentioned enzymesin leucine catabolism pathway, gene targets for metabolic engineering ofthe CHO cell line A that will suppress the biosynthesis of isovalericacid were identified.

Materials and Methods:

Mutation Analysis

Genomic DNA sequence flanking the translated regions of Ivd, Mccc1 andMccc2 (at the 5′ and 3′ ends) was identified and used as a template forPCR primer design using the Primer3 algorithm . RNA was prepared fromCHO cell line A using the Qiagen RNeasy Kit (Qiagen) which was in turnused as a template for oligo dT primed cDNA synthesis using theSuperScript III First-Strand Synthesis System for RT-PCR (LifeTechnologies). An extension temperature and magnesium concentrationoptimized PCR reaction was performed using Pfu Turbo HotStart 2X MasterMix (Agilent). The samples were purified using the QIAquick PCRPurification Kit (Qiagen) and sent for sequence analysis at Wyzerbio(Cambridge, Mass.).

Results:

The C_(T) and ΔC_(T) values for the genes in the leucine pathway areshown in FIG. 25 . The gene expression data from thephenylalanine/tyrosine pathway indicates that CHO cell line A has lowerlevels of AUH enzyme relative to the other enzymes in the pathway.However, this data doesn't strongly explain the high levels ofisovaleric acid production as reported in the earlier experiments, asthe enzyme is further downstream of the node which branches out towardsisovaleric acid production (FIG. 25 ). Therefore, loss of function(enzyme activity) of IVD, MCCC1 and/or MCCC2 could give a betterrationale for isovaleric acid production. DNA sequencing results revealthe mutation status and the likely level of function of the encodedenzyme. Should an enzyme activity altering mutation in the IVD, MCCC1,and MCCC2 genes be found, a metabolic engineering approach that entailsoverexpression of the corresponding wild type (non-mutated) gene will beundertaken, in this case the gene origin would be from a CHO cell linealthough other origin sources for wild type genes are obtainable andcould be used such as human, rat, mouse. In addition to this, AUH genewill also be overexpressed in the same CHO cell line A.

A correlation of the gene acronyms with the full nomenclature for thegenes discussed herein is provide below in Table 5.

TABLE 5 Genes of the phenylanaline/tyrosine and leucine pathways as alsoillustrated in FIGS. 24 and 25 Symbol Name Pah phenylalanine hydroxylaseMif macrophage migration inhibitory factor Got1 glutamic-oxaloacetictransaminase 1, soluble Got2 glutamatic-oxaloacetic transaminase 2,mitochondrial Hpd 4-hydroxyphenylpyruvic acid dioxygenase Hgdhomogentisate 1, 2-dioxygenase Gstz1 glutathione transferase zeta 1(maleylacetoacetate isomerase) Fah fumarylacetoacetate hydrolase Bcat1branched chain aminotransferase 1, cytosolic Bckdha branched chainketoacid dehydrogenase E1, alpha polypeptide Bckdhb branched chainketoacid dehydrogenase E1, beta polypeptide Dbt dihydrolipoamidebranched chain transacylase E2 Dld dihydrolipoamide dehydrogenase Ivdisovaleryl coenzyme A dehydrogenase Acadm acyl-Coenzyme A dehydrogenase,medium chain Mccc1 methylcrotonoyl-Coenzyme A carboxylase 1 (alpha)Mccc2 methylcrotonoyl-Coenzyme A carboxylase 2 (beta) Auh AU RNA bindingprotein/enoyl-coenzyme A hydratase Hmgcl3-hydroxy-3-methylglutaryl-Coenzyme A lyase Fasn fatty acid synthaseNup62- nucleoporin 62-interleukin 4 induced 1/L-amino acid il4i1 oxidase

Example 13: Experiment to Ectopically Express in CHO Cell Line a, theMouse Genes of HPD, HGD, PAH, AUH and Wild Type Form of any Other GeneTarget Determined in Example 12 Through Mutation Analysis

Goal:

Based on low gene expression levels relative to Beta Actin as determinedby the RT qPCR assay, four targets were selected for overexpression inCHO cells: Auh (leucine metabolic pathway) and HPD, HGD, and PAH(phenylalanine/tyrosine metabolic pathway). In addition, based on theloss of activity mutation status of IVD, MCCC1 and MCCC2, the wild typegene of the mutated enzymes will be selected for overexpression in CHOcells. Generally where genes are found to be mutated or inactive thestrategy is to overexpress the wild type genes. The goal is to clonemouse mRNA sequences of these genes into a commercially availablemammalian expression vector and transfect CHO cell line A with theseexpression vectors. The resulting cell lines have a high level ofexpression of the mouse AUH, HPD, HGD, or PAH gene and by extension, ahigher level of AUH, HPD, HGD, or PAH enzyme activity. Increasedactivity in these key enzymes will likely improve metabolic flux andreduce the cellular concentration of certain known inhibitorysubstances.

Materials and Methods:

Overexpression cassettes were constructed using mouse cDNA sequencesfrom the MGC collection of AUH, HPD, HGD, and/or PAH genes. Thesequences were provided as E. coli glycerol stocks containing shuttlevectors with cDNAs of the target genes (GE Dharmacon). PCR primers weredesigned using the Primer3 algorithm to amplify the coding regions ofthe cDNAs in reactions with a proof-reading polymerase called Pfu TurboHotStart 2X Master Mix (Agilent). The PCR products were directionallycloned into the pcDNA Gateway Direction TOPO Expression vector(Invitrogen). The vector contains a viral promoter sequence (Humancytomegalovirus immediate early promoter), a directional TOPO cloningsite, an epitope tag (V5) for detection using anti-V5 antibodies, apolyadenylation sequence (Herpes Simplex Virus thymidine kinase), andantibiotic resistance gene (Blasticidin). The vectors are sequenceconfirmed by WyzerBio and transfected into CHO Cell Line A for overexpression using the GenePulser XCell eletroporator (BioRad) andrecovered in the presence of blasticidin for selective pressure. Theengineered CHO cells are assessed for expression of the trans-gene by RTqPCR, western hybridization, and enzyme assay. Upon selection withmedium containing blasticidin the cells are cryopreserved for laterexperimentation in bioreactors.

The transfected cell lines are therefore engineered to express mouseAUH, HPD, HGD, and/or PAH genes at high levels and by extension, haveincreased AUH, HPD, HGD, or PAH enzymatic activity. Analysis by RT qPCR,Western hybridization, and enzyme assay is performed to reveal increasein the total Auh, Hpd, Hgd, or Pah gene expression and enzyme activity(both endogenous and transgenic) in the newly created transgenic celllines.

Example 14: Experiment to Probe the Suppression of Inhibitor Formationin Genetically Engineered Cell Lines that Expresses the Target GenesDetermined in Examples 11 and 12

Goal:

As part of this experiment, the phenotype of the clones (and pools)overexpressing the target genes was investigated. Phenotypedetermination mainly entails the peak cell densities and productivity ofthese clones in HIPDOG fed-batch culture and/or the level of inhibitormolecule accumulation in the culture.

Materials and Methods:

Cells and Bioreactor Setup

CHO cells used in the experiment are the engineered form of CHO cellline A obtained from overexpression of the target genes PAH, HPD, HGD,AUH and wild type form of any other gene target determined in Example 11and 12 by mutation analysis. Two conditions are be tested as part ofthis experiment: A) HIPDOG fed-batch culture using the CHO cellsexpression all the target genes and B) HIPDOG fed-batch culture with CHOcells expressing GFP protein. Details on the use HIPDOG technology aredescribed in the material and methods section of Example 1.Exponentially growing cells from seed culture are inoculated at 1×10⁶cells/mL into production bioreactors that employes HiPDOG process. Forboth the conditions, HiPDOG control is in operation between day 2 andday 7 of the culture. Viable cell density, lactate, ammonia and aminoacid concentrations are measured on daily basis until day 12 (aminoacids measured only for first seven days). For both conditions, theinoculum viable cell density targeted (1×10⁶ cells/mL), and the culturevolume (1 L) and the process parameters including the temperature (36.5C), pH (6.9-7.2) and the agitation rate (267 rpm) were identical. Forboth conditions, the base media used will be Medium A and the feedmedium is Medium B. Supernatant samples from various conditions acrossboth the experiments are analyzed for the levels of the newly identifiedinhibitors using the NMR technology described in the Materials andMethods section of Example 1. Amino acid concentrations are measured forsamples using UPLC based amino acid method which is described in detailbelow.

Amino Acid Analysis

10 μL of either a standard amino acid mix solution or a spent mediasample (10 times diluted sample) is mixed with 70 μL of AccQ●Tag Ultraborate buffer (Waters UPLC AAA H-Class Applications Kit [176002983]),and 20 μL of AccQ●Tag reagent previously dissolved in 1.0 mL of AccQ●TagUltra reagent diluent was added. The reaction is allowed to proceed for10 min at 55° C. Liquid chromatographic analysis was performed on aWaters Acquity UPLC system, equipped with a binary solvent manager, anautosampler, a column heater and a PDA detector. The separation columnis a Waters AccQ●Tag Ultra column (2.1 mm i.d.×100 mm, 1.7 μmparticles). The column heater is set at 55° C., and the mobile phaseflow rate was maintained at 0.7 mL/min. Eluent A is 10% AccQ●Tag Ultraconcentrate solvent A, and eluent B is 100% AccQ●Tag Ultra solvent B.The nonlinear separation gradient is 0-0.54 min (99.9% A), 5.74 min(90.0% A), 7.74 min (78.8% A), 8.04-8.64 min (40.4% A), 8.73-10 min(99.9% A). One microliter of sample is injected for analysis. The PDAdetector is set at 260 nm. The previously determined elution times forthe amino acids are used to identify the specific amino acid peaks onthe chromatogram for each sample. The amino acid concentrations areestimated using the area under the peak and the calibration curvegenerated using the standard solution (Amino Acids Standard H, ThermoScientific, PI-20088).

Results:

Cells expressing the target gene channel lower carbon (phenylalanine,tyrosine and leucine) towards biosynthesis of inhibitor resulting inlower accumulations of the compounds in the culture medium. However, thecells expressing the GFP molecule (negative control) channel more carbontowards inhibitor production leading to higher accumulations of the samein the culture media. Such differences in the levels of inhibitorproduction and accumulation results in growth differences leading tohigher peak cell densities in the cells expressing the target gene setcompared to cell expressing GFP. Such increase in the total cells alsoleads to higher over yield of the recombinant protein produced by thecells.

Example 15: Experiment to Determine the Gene Targets for MetabolicEngineering for Suppressing the Biosynthesis of the Inhibitors Relatedto Phenylalanine/Tyrosine Pathway

Goal

This experiment was performed to determine the cause for production ofinhibitor molecules including phenyllactate, 4-hydroxyphenylpyruvate and3-(4-hydroxyphenyl)lactate. Gene expression of all the enzymes in thephenylalanine/tyrosine pathway was probed using Real Time QuantitativePolymerase Chain Reaction (RT-qPCR) assay. Based on the gene expressionand the biochemistry of the phenylalanine/tyrosine catabolic pathway,gene targets for metabolic engineering of the CHO cells (CHO cell lineA, CHO cell line B and parental cell line) that suppress thebiosynthesis of above mentioned inhibitors, were identified.

Materials and Methods

Gene Expression Analysis

RT-qPCR assay was used to assess relative gene expression levels ofenzymes in the phenylalanine/tyrosine metabolic pathways. RT qPCRmeasures transcript abundance, and hence, gene expression by amplifyinga target cDNA sequence using PCR in combination with a detection reagent(i.e. SYBR Green). SYBR green is a molecule that fluoresces when boundto double stranded DNA and the fluorescence can be measured in real timeduring the RT qPCR assay. The amount of fluorescence is directlyproportional to the amount of double stranded PCR product (also calledamplicon) in the reaction. Relative gene expression levels aredetermined by measuring the number of PCR cycles required for SYBR greenfluorescence to surpass the background fluorescence and increaselogarithmically. This cycle number is commonly referred to as the C_(T)(Threshold Cycle). A transcript in high abundance would have a lowerC_(T) value as it would require fewer PCR cycles for the fluorescence tosurpass the background fluorescence where, conversely, a transcript inlower abundance would have a higher C_(T) value as it would require morePCR cycles for the fluorescence to surpass the background level.

The RT qPCR assay was performed using an Applied Biosystems 7500 RealTime PCR system (Applied Biosystems) and the PowerUP SYBR Green MasterMix reagent (Life Technologies). The PCR primers were designed using thePrimer3 algorithm based on genomic DNA sequences contained in theChinese Hamster Ovary (CHO) genome browser. RNA was prepared from CHOcell line A using the Qiagen RNeasy Kit (Qiagen) which was in turn usedas template for oligo dT primed cDNA synthesis using the SuperScript IIIFirst-Strand Synthesis System for RT-PCR (Life Technologies). The C_(T)values of the targeted metabolic genes were tabulated and compared tothe C_(T) value of a well characterized housekeeping gene, beta-Actin(B-Actin). The difference between the C_(T) of the target gene and theC_(T) of B-Actin was reported as the ΔC_(T). High ΔC_(T) value indicateslow gene expression level.

Results

The C_(T) and ΔC_(T) values for the genes in the phenylalanine/tyrosinepathway for CHO cell line A and parental cell line are shown in FIG. 26. CHO cell line B has similar level of gene expression as CHO cell lineA (data not shown). The gene expression data from thephenylalanine/tyrosine pathway indicates that CHO cell line A andparental cell line have low expression of the PAH, HPD and HGD genes.The protein (or enzyme) levels correlate to the transcript levels, lowlevels of PAH enzyme can result in the phenylalanine being converted tophenylpyruvate and subsequently to phenyllactate by catalytic action ofGOT1/GOT2 and MIF enzymes (or non-specific enzymatic reactions), both ofwhich are expressed at high levels in CHO cell line A and parental cellline.

Similarly, due to low levels of HPD and HGD enzymes in CHO cell line Aand parental cell line, tyrosine is channeled towards production of4-hydroxyphenylpyruvate and 3-(4-hydroxyphenyl)lactate by catalyticaction of GOT1/GOT2 and MIF enzymes (or non-specific enzymaticreactions). One way to reduce the channeling of the phenylalanine andtyrosine flux towards inhibitor production would be to down-regulate theexpression of GOT1/GOT2 and MIF genes. However, GOT1/GOT2 and MIF arecritical for other physiologically important metabolic functions.Therefore, down-regulation or knocking-out of these two enzymes wouldn'tpotentially yield a viable cell line. An alternative way to reduceinhibitor biosynthesis is through overexpression of the PAH, HPD and HGDgenes which would then channel the flux away from inhibitor productionand towards production of Krebs cycle metabolites (which are used forenergy synthesis in mitochondria). Hence, the metabolic targets chosenfor phenylalanine/tyrosine pathway are PAH, HPD and HGD genes.

In addition to the three targets in the phenylalanine/tyrosine pathwayidentified by RT qPCR (PAH, HGD, and HPD), a fourth target was examined.The PAH enzyme requires a cofactor called tetrahydrobiopterin (BH₄) forits catalytic activity. Mammalian cells synthesize BH₄ using GTP as asubstrate (FIG. 27A). The enzymes involved in the biosynthesis of BH₄include GCH1, PTS and SPR. Further, BH₄ is converted toBH₄-4a-carbinolamine during the reaction catalyzed by PAH activity,which is recycled back to BH₄ by the activity of PCBD1 and QDPR enzymes.The expression of genes encoding for the biosynthesis and the recyclingenzymes was likewise assayed by RT qPCR in CHO cells (cells used forgene expression analysis were those which went through first round oftransfection with mouse PAH gene; see Example 16) (FIG. 27B). Based onthe high C_(T) value, high ΔC_(T), and hence, low level of geneexpression, the PCBD1 gene was also selected for overexpression. Allother genes in the BH₄ pathway were expressed at reasonable levels (seeFIG. 27B).

Example 16: Experiment to Overexpress PAH, HPD, HGD and PCBD1 in CHOCell Lines

Goal

Based on low gene expression levels relative to B-Actin, as determinedby the RT qPCR assay, four targets were selected for overexpression inCHO cells: HPD, HGD, PAH and PCBD1. The main goal of this experiment wasto clone mRNA sequences of these genes into a commercially availablemammalian expression vectors and transfect CHO parental cell line, CHOcell line A and CHO cell line B with the expression vectors. Theresulting cells were expected to have a high level of expression of HPD,HGD, PAH and PCBD1 genes and by extension, a higher level of HPD, HGD,PAH and PCBD1 enzyme activity. Increased activity of these key enzymeshave improved metabolic flux and reduced the cellular concentration ofthe identified inhibitory substances.

Materials and Methods

Expression vectors for HPD, HGD, PAH and PCBD1 genes were constructedusing mouse cDNA sequences from the MGC collection (FIG. 28 ). Thesequences were provided by GE Dharmacon as E. coli glycerol stockscontaining shuttle vectors with cDNAs of the target genes. PCR primerswere designed using the Primer3 algorithm to amplify the coding regionsof the cDNAs in reactions with a proof-reading polymerase called PfuTurbo HotStart 2X Master Mix (Agilent). The PCR products were clonedinto commercially available constitutive expression vectors withdifferent antibiotic resistance genes to allow for individual selectionof the expression plasm ids shown in Table 6. In addition, controlvectors were also provided by the vendor to serve as a negative control(transfection control). The vectors were sequence confirmed byWyzerBiosciences (Cambridge, Mass.). The expression and control plasmids were transfected into CHO parental cells, CHO cell line A and CHOcell line B using the GenePulser XCell eletroporator (BioRad) andrecovered in the presence of antibiotics for selective pressure. Astepwise approach was taken whereby the cells were first transfectedwith the PAH expression vector and recovered. The PAH expressing cellpools were then transfected with HPD and HGD together, and the resultantcell pool was transfected with PCBD1 until the final product, a 4-timestransfected cell line, was achieved. Upon selection with mediumcontaining antibiotics, the cell pools were cryopreserved for laterexperimentation in bioreactors. FIG. 28 shows the expressed mouse genes,the expression vectors created in Pfizer's laboratories and theantibiotic selection gene. CHO cell line A was only transfected with PAHand was not taken through subsequent set of transfections. Table 6 showsa summary of the plasm ids used for each gene expressed, including thecommercial expression vector used, the antibiotic used as selectionpressure, and the vendor of the expression vector. Also listed are thenull vectors used to generate negative transfection control cell lines.

TABLE 6 Vendor of Gene Vector for Transfection Antibiotic SelectionExpression Vector PAH pcDNA3.2/V5/GW/D- G418 (Geneticin) Invitrogen TOPO(Thermo Fisher) HPD pMONO-hygro-mcs Hygromycin Invivogen HGDpcDNA6.2/V5/GW/D- Blasticidin Invitrogen TOPO (Thermo Fisher) PCBD1pSELECT-puro-mcs Puromycin Invivogen Control for PAH pcDNA3.2/V5/GW-CATG418 (Geneticin) Invitrogen (Thermo Fisher) Control for HPDpMONO-hygro-mcs Hygromycin Invivogen Control for HGD pcDNA6.2/V5/GW-CATBlasticidin Invitrogen (Thermo Fisher) Control for PCBD1pSELECT-puro-mcs Puromycin InvivogenResults

Cell pools were generated after transfection with PAH, HPD, HGD, andPCBD1 expression vectors (called 4x-tfxn cell pools) along with theaccompanying negative control vectors (either empty vector or vectorcontaining chloramphenicol acetyltransferase (CAT)) (called4x-control-tfxn cell pools). 4x-tfxn cell pools obtained fromtransfection were designed to express mouse PAH, HPD, HGD and PCBD1genes at high levels and have increased PAH, HPD, HGD and PCBD1enzymatic activity. Analysis by RT qPCR (see Example 17), and westernhybridization (data not shown) was used to determine changes in PAH,HPD, HGD and PCBD1 gene expression (both endogenous and transgenic) inthe newly created transgenic cell pools.

Example 17: Experiment to Probe the Expression of Mouse Transgenes (PAH,HPD, HGD and PCBD1) in the Cell Pools Generated from the QuadrupleTransfections

Goal

An RT qPCR assay was performed to assess relative gene expression levelsof mouse transgenes PAH, HPD, HGD, and PCBD1 in 4x-tfxn cell poolsderived from parental cell line or CHO cell line B.

Materials and Methods

The RT qPCR assay was performed using an Applied Biosystems 7500 RealTime PCR system (Applied Biosystems) and the PowerUP SYBR Green MasterMix reagent (Life Technologies). The PCR primers were designed using thePrimer3 algorithm based on the mouse cDNA sequences from the MGCcollection provided by GE Dharmacon. RNA was prepared from the 4x-tfxncells pools and 4x-control-tfxn cell pools of CHO cell line B orparental cell line using the Qiagen RNeasy Kit (Qiagen), which was inturn used as template for oligo dT primed cDNA synthesis using theSuperScript III First-Strand Synthesis System for RT-PCR (LifeTechnologies). The C_(T) values of the mouse PAH, HPD, HGD, and PCBD1genes were tabulated and compared to the C_(T) value of a wellcharacterized housekeeping gene, B-Actin. The delta between the C_(T) ofthe target gene and the C_(T) of B-Actin was reported as the ΔC_(T).Successful expression of mouse transgenes PAH, HPD, HGD and PCBD1 genesin 4x-tfxn cell pools was identified by reduced ΔC_(T) value (and hence,increased gene expression level) when compared to the ΔC_(T) value ofthe transgenes in the negative control (4x-control-tfxn) cell pools,which lacks mouse PAH, HPD, HGD and PCBD1 expression.

Results

The C_(T) values for mouse PAH, HPD, HGD and PCBD1 genes in 4x-tfxn cellpools and the corresponding negative control pools (4x-control-tfxn) areshown in Table 7. Lower ΔC_(T) values were observed for the mousetransgenes in the 4x-tfxn cell pools of CHO cell line B and parentalcell line compared to the corresponding negative control cell pools(4x-control-tfxn). This indicates that the gene expression of thetargets was higher in 4x-tfxn pools and therefore promotes thechanneling of amino acid flux away from the phenyllactate,3-(4-hydroxyphenyl)lactate and 4-hydroxyphenyllactate inhibitorbiosynthesis pathway (see Example 19). The apparent presence ofexpression of mouse PAH, HPD, HGD and PCBD1 in 4x-control-tfxn cellpools is due in part to high levels of sequence homology between thetransgenic (mouse) and endogenous (hamster) PAH, HPD, HGD and PCBD1genes. This suggests that the data reflect accurately the total level ofPAH, HPD, HGD and PCBD1 gene expression inclusive of both transgenic andendogenous genes.

Table 7 shows the expression levels, as assayed by RT-qPCR, of mousePAH, HPD, HGD and PCBD1 genes in quadruple transfected (4x-tfxn) ornegative quadruple transfection control (4x-control-tfxn) cell pools ofCHO parental cell line and CHO cell line B.

TABLE 7 Average ΔC_(t) Compared to B-Actin (Low ΔC_(t) value = HighExpression) CHO Parental CHO Parental CHO CHO Cell Line Cell Line CellLine B Cell Line B Gene 4x-control-tfxn 4x-tfxn 4x-control-tfxn 4x-tfxnPAH 11.53 8.2 11.96 7.86 HPD 18.13 5.02 18.01 3.11 HGD 20.21 16.34 19.2315.25 PCBD1 12.7 6.24 14.66 3.97

Example 18: Probing the Ability of Cell Pools that Express the FourTransfected Mouse Genes Including PAH, HPD, HGD and PCBD1 (4x-Tfxn) toGrow in Tyrosine-Free Medium

Goal

Expression of PAH and PCBD1 enzyme activity conferred the ability tocells to catalyze the synthesis of tyrosine from phenylalanine andtherefore to promote the channeling of amino acid flux away from thephenyllactate, 3-(4-hydroxyphenyl)lactate and 4-hydroxyphenyllactateinhibitor biosynthesis pathway (see Example 19). The goal of thisexperiment was to test if 4x-tfxn cell pools expressing the four mousegenes including PAH, HPD, HGD and PCBD1, derived from CHO cell line B orparental host cell line, have the ability to proliferate intyrosine-free medium conditions.

Materials and Methods

Cells, Medium and Experiment Setup

4x-tfxn cell pools or 4x-control-tfxn cell pools of CHO cell line B orparental cell line were spun down and cell pellets were inoculated inMedium D (Medium C without tyrosine, but with supplementation ofadditional amount (2 mM) of phenylalanine). Medium C is approximately athird in concentration of various amino acids of the levels in Medium A.Medium A is a fortified version of insulin-free Medium 9 as disclosed inU.S. Pat. No. 7,294,484, table 14, with slight differences inconcentrations of sodium bicarbonate and potassium chloride, andcontaining Pluronic F68 instead of polyvinyl alcohol. It was fortifiedby adding 10% glutamine-free Medium 5 (U.S. Pat. No. 7,294,484, table7), and by further raising the concentrations of eight amino acids (Glu,Tyr, Gly, Phe, Pro, Thr, Trp and Val). The concentrations of amino acidsare listed in the Table 8 below.

TABLE 8 Concentration of Amino Acids in Medium A Amino AcidsConcentration in Medium A (mM) alanine 0.4 arginine 5.3 asparagine•H2O21.1 aspartic acid 2.3 cysteine•HCl•H2O 0.4 cystine•2HCl 1.5 glutamicacid 0 monosodium glutamate 2.0 glutamine 0 glycine 3.6histidine•HCl•H2O 2.7 isoleucine 5.4 leucine 9.4 lysine•HCl 8.9methionine 3.1 phenylalanine 4.5 proline 9.1 serine 11.8 threonine 10.8tryptophan 2.3 tyrosine•2Na•2H2O 5.1 valine 10.3

Cells were inoculated at 0.3E6 cells/mL in 25 mL working volume in 125mL shake flasks. Shake flasks were incubated on a shaking platform (125rpm) at 36.5 C and 5% carbon dioxide environment. Cell growth wasmonitored for 3 days. On day 3, cells were spun down and the cellpellets were re-inoculated into fresh Medium D at 0.3E6 cells/mL andcell growth was monitored for next 5 days.

Results

In the first passage, over the course of the three days, both the cellline B and parental cell line derived 4x-tfxn cell pools grew well intyrosine-free media conditions whereas the 4x-control-tfxn cell poolsdidn't show any proliferation (FIG. 29 ). In the second passage, similarto the first round, 4x-tfxn cell pools grew well whereas the4x-control-tfxn cell pools didn't show any proliferation. This datasuggested that 4x-tfxn cell pools were able to synthesize tyrosine fromphenylalanine and hence promote the channeling of amino acid flux awayfrom the phenyllactate, 3-(4-hydroxyphenyl)lactate and4-hydroxyphenyllactate inhibitor biosynthesis pathway (see Example 19).

Further, it was also observed that 3x-tfxn cell pools derived from boththe cell line B and parental cell line (with overexpression of PAH, HPDand HGD) were unable to proliferate in tyrosine-free medium (data notshown). This suggested that expression of PCBD1 was critical forcellular ability to biosynthesize tyrosine. It has been previouslyreported that activity of PAH enzyme is enhanced at high pH (Parniak etal., 1988). Culturing the 4x-tfxn cell pools in tyrosine-free conditionsat higher pH (>7.0) further increased the cellular growth rate andviability (data not shown).

Example 19: Demonstrating (i) Similar or Better Growth/Productivity of4x-Tfnx Cell Pools in Tyrosine-Free Fed-Batch Cultures when Compared4x-Control-Tfxn Cell Pools in Tyrosine Supplemented Cultures and, (ii)Reduced Accumulation of 3-(4-Hydroxyphenyl)Lactate, a Byproduct ofTyrosine Pathway, in 4x-Tfxn Cultures

Goal

The goal of this experiment was to demonstrate that 4x-tfxn cell poolscan grow in tyrosine-free HiPDOG cultures at similar or higher growthrates and reach similar or higher peak cell densities and producesimilar or higher titers, when compared to 4x-control-tfxn cell pools intypical (tyrosine-supplemented) HiPDOG cultures. Another goal was toestablish that the combination of PAH, HPD, HGD and PCBD1 expressionalong with cultivation in tyrosine-free media suppresses the productionof the metabolite byproduct of tyrosine pathway,3-(4-hydroxyphenyl)lactate.

Materials and Methods:

Cells and Bioreactor Setup

4x-tfxn cells and 4x-control-tfxn cell pools derived from the CHO cellline B, expressing a recombinant antibody, were used in this example.4x-tfxn cell pools expressed the four mouse enzymes (PAH, HPD, HGD andPCBD1), whereas the 4x-control-tfxn cell pools are the control cellsthat expressed only the resistance marker for the selection pressure.

Cell line B derived 4x-tfnx cell pools or the 4x-control-tfnx cell poolswere inoculated at 1.2E6 cells/mL in HiPDOG fed-batch cultures usingtyrosine-free Medium A or original formulation of Medium A (containingtyrosine), respectively (for information on Medium A, see Example 18).To supplement nutrients during the culture, tyrosine-free Feed Medium Bwas used for 4x-tfxn cell pool fed-batch culture whereas the originalcomposition of Feed Medium B (containing tyrosine) was used for4x-control-tfxn fed-batch cultures (for information on Medium B, seebelow). For both the conditions, the culture volume (1 L) and theprocess parameters including the temperature (36.5 C), pH (until day 2:7.15-7.20, from day 2 onwards: 7.10-7.15) and the agitation rate (259rpm) were identical. HiPDOG strategy was employed throughout the run.Viable cell density, glucose, lactate, ammonia and amino acidconcentrations were measured on daily basis until day 12. Supernatantsamples from both the conditions were analyzed for the levels of the3-(4-hydroxyphenyl)lactate using the NMR technology described below.Culture amino acid levels were measured using amino acid analysis methoddescribed below.

Medium B has the same composition as Medium 5 (U.S. Pat. No. 7,294,484,table 7), but with higher levels of the amino acids (by a factor of2.5). The concentrations of amino acids in Medium B are shown in Table9.

TABLE 9 Concentration of Amino Acids in Medium B Amino AcidsConcnetration in Medium B (mM) alanine 6.0 arginine 32.9 asparagine•H₂O54.0 aspartic acid 15.0 cysteine•HCl•H₂O 0.0 cystine•2HCl 4.7 glutamicacid 6.0 monosodium glutamate 0.0 glutamine 0.0 glycine 6.0histidine•HCl•H₂O 10.5 isoleucine 27.0 leucine 38.9 lysine•HCl 30.0methionine 12.0 phenylalanine 15.0 proline 18.0 serine 45.2 threonine24.0 tryptophan 4.8 tyrosine•2Na•2H₂O 12.0 valine 24.0NMR Sample Preparation, Data Acquisition and Processing

1000 μL of each sample was filtered using Nanosep 3K Omegamicrocentrifuge filter tubes for 60 minutes, and 630 μL of the filteredsample was used for NMR analysis. These filters are preserved withglycerol, and as such some trace amounts of glycerol may appear in theanalysis. Internal standard solution was added to each sample solution,and the resulting mixture was vortexed for 30 s. 700 μL of thecentrifuged solution was transferred to an NMR tube for dataacquisition.

NMR spectra were acquired on a Varian four-channel VNMRS 700 MHz NMRspectrometer equipped with a cryogenically cooled 1H/13C tripleresonance biomolecular probe with auto tuning. The pulse sequence usedwas a 1 D-tnnoesy with a 990 ms presaturation on water and a 4 sacquisition time. Spectra were collected with 32 transients and 4steady-state scans at 298 K.

Spectra were processed and .cnx files were generated using the Processormodule in Chenomx NMR Suite 8.0. Compounds were identified andquantified using the Profiler module in Chenomx NMR Suite 8.0 with theChenomx Compound Library version 9, containing 332 compounds. Forreporting purposes, the profiled concentrations have been corrected toreflect the composition of the original sample, instead of the contentsof the NMR tube. During sample preparation, each sample is diluted byintroducing an internal standard and, where necessary, to increase theanalyzed volume of a small sample.

Amino Acid Analysis

10 μL of either a standard amino acid mix solution or a spent mediasample (10 times diluted sample) was mixed with 70 μL of AccQ●Tag Ultraborate buffer (Waters UPLC AAA H-Class Applications Kit [176002983]),and 20 μL of AccQ●Tag reagent previously dissolved in 1.0 mL of AccQ●TagUltra reagent diluent was added. The reaction was allowed to proceed for10 min at 55° C. Liquid chromatographic analysis was performed on aWaters Acquity UPLC system, equipped with a binary solvent manager, anautosampler, a column heater and a PDA detector. The separation columnwas a Waters AccQ●Tag Ultra column (2.1 mm i.d.×100 mm, 1.7 μmparticles). The column heater was set at 55° C., and the mobile phaseflow rate was maintained at 0.7 mL/min. Eluent A was 10% AccQ●Tag Ultraconcentrate solvent A, and eluent B was 100% AccQ●Tag Ultra solvent B.The nonlinear separation gradient was 0-0.54 min (99.9% A), 5.74 min(90.0% A), 7.74 min (78.8% A), 8.04-8.64 min (40.4% A), 8.73-10 min(99.9% A). One microliter of sample was injected for analysis. The PDAdetector was set at 260 nm. The previously determined elution times forthe amino acids are used to identify the specific amino acid peaks onthe chromatogram for each sample. The amino acid concentrations wereestimated using the area under the peak and the calibration curvegenerated using the standard solution (Amino Acids Standard H, ThermoScientific, PI-20088).

Results

Cell pools in both the cultures grew in an exponential fashion withslightly better growth observed in the 4x-tfxn cell tyrosine-freefed-batch culture as compared to 4x-control-tfxn culture (FIG. 30A).Both cultures attained peak cell densities of 35E6 cells/mL between days7 and 9 of the cultures. In both the cultures, the cell viabilities weremore than 97% through day 12 of the culture (FIG. 30B). Titer profilefor both the cultures were very similar with day 11 titer at 2.5 g/L(FIG. 30C). Further, it was observed that 4x-tfxn cells consumedphenylalanine at a faster rate than the 4x-control-tfxn cells (data notshown). Tyrosine accumulation was observed in 4x-tfxn cultures (data notshown). This suggested that 4x-tfxn cells converted phenylalanine totyrosine to supply the same for their physiological needs (biomass andprotein synthesis). Excess tyrosine was secreted into the culture.4x-control-tfxn culture was supplemented with exogenous tyrosine ininoculation and feed media. The levels of tyrosine decreased over thecourse of the 4x-control-tfxn culture indicating consumption of tyrosineby these cells (data not shown). In addition, the levels of a byproductof tyrosine metabolism, 3-(4-hydroxyphenyl)lactate, were significantlylower in 4x-tfxn culture as compared to 4x-control-tfxn cultureindicating lower channeling of phenylalanine and/or tyrosine towardsbyproduct synthesis (FIG. 30D).

Example 20: Probing the Effect of 2-Methylbutyrate and Isobutyrate onGrowth of CHO Cells

Goal:

2-methylbutyrate and isobutyrate are byproducts of isoleucine and valinepathways, which were observed to accumulate in the HiPDOG fed-batchcultures of CHO cell line A. The level of accumulation on day 7 wereabout 9 mM for 2-methylbutyrate and 2 mM for isobutyrate (data notshown; accumulation for CHO cell line C are in the same order (seeExample 21)). This experiment was setup to probe the effect of these twocompounds individually on growth of CHO cell line A in the concentrationrange observed in the HiPDOG fed-batch cultures.

Materials and Methods:

CHO cell line A producing a recombinant antibody were inoculated at lowcell densities (0.1E6 cells/mL) in various conditions in triplicates in6-well plate cultures. The working volume for each well on day 0 was 4mL. The conditions tested include fresh Medium A or fresh Medium Asupplemented individually with 2-methylbutyrate or isobutyrate atvarious concentrations. The concentrations tested for 2-methylbutyrateare [0, 5, 10 and 20 mM] and for isobutyrate are [0, 1, 2 and 4 mM]. The6-well plates were incubated on a shaking platform in 36.5 C and 5%carbon dioxide. Cell growth in all the conditions was monitored for 5days.

Results:

FIG. 31 shows the independent effect of the 2-methylbutyrate orisobutyrate on growth of the CHO cell line A. Cells cultured in freshmedium grew very well. Cell growth was suppressed when cells werecultured in fresh media supplemented with 2-methylbutyrate (FIG. 31A) orisobutyrate (FIG. 31B) at concentrations higher than 5 mM or 1 mM,respectively. This demonstrates that 2-methylbutyrate and isobutyrateinhibit cell growth. 2-methylbutyrate is a metabolic byproduct ofisoleucine metabolism and isobutyrate is a metabolic byproduct of valinemetabolism.

Example 21: Demonstrating the Reduction in the Accumulation of2-Methylbutyrate or Isobutyrate Through Limitation of Isoleucine orValine, Respectively, in Fed-Batch Cultures of CHO Cells (CHO Cell LineC)

Goal

The main goal of this example was to demonstrate reduction in theaccumulation of the 2-methylbutyrate or isobutyrate in fed-batchcultures of CHO cells by limiting the supply of the isoleucine orvaline, respectively.

Materials and Methods

Cells and Bioreactor Setup

The GS-CHO cell line (cell line C) expressing a recombinant antibody wasused in this example. Two conditions were tested as part of the thisexample: A) fed-batch culture with low levels of isoleucine and valine(Low AA), B) fed-batch cultures with normal amino acids concentrations(Control). The experiment was carried out for 12 days.

Exponentially growing cells from seed cultures were inoculated at about4E6 cells/mL into production bioreactors that employed a typicalfed-batch process (with typical levels of amino acids) or the low aminoacid fed-batch process. In the low amino acid conditions, theconcentrations of isoleucine and valine were maintained between 0.5 mMand 1 mM for first seven days of the culture after which they wereallowed to increase beyond 1 mM. Viable cell density, glucose, lactate,ammonia and amino acid concentrations were measured on daily basis untilday 12. For both the conditions, the culture volume (1 L) and theprocess parameters including the temperature (36.5 C), pH (6.9-7.2) andthe agitation rate (259 rpm) were identical. The base media used incontrol condition was Medium A and that used in low amino acidconditions was the modified version of Medium A with low concentrationsof amino acids, including tyrosine, phenylalanine, methionine,tryptophan, serine, glycine, threonine, leucine, isoleucine and valine.The feed medium used for both the conditions was Medium B. Amino acidlevels in Medium B were adjusted such that the amount of amino aciddelivered through feeding Medium B, in a semi-continuous fashion, isapproximately equal to the amount of amino acid taken up by the culture.The feed rate used was proportional to integral viable cells in theculture. Supernatant samples from both the conditions were analyzed forthe levels of the 2-methylbutyrate and isobutyrate using the NMRtechnology described in example 19. Culture amino acid levels weremeasured using amino acid analysis method described in example 19.

Results

The concentrations of the amino acids, isoleucine and valine, weresuccessfully maintained between 0.5 mM-1 mM in the Low AA condition(FIGS. 32A and 32C). Such limitation of amino acid levels in the Low AAcondition resulted in lower accumulation of 2-methylbutyrate (FIG. 32B)and isobutyrate (FIG. 32D).

Example 22: Experiment to Determine the Gene Targets for MetabolicEngineering for Suppressing the Biosynthesis of the Inhibitors(Isovalerate, 2-Methylbutyrate and Isobutyrate) Produced from theBranched Chain Amino Acid Pathway

Goal

This experiment was performed to determine the cause for thebiosynthesis of isovalerate, 2-methylbutyrate and isobutyrate, which arebyproducts of branched chain amino acid metabolism that accumulated tovery high levels in fed-batch cultures of CHO cells. Gene expression ofall the enzymes in the branched chain amino acid pathway (BCAA) wasprobed. Based on the gene expression analysis in BCAA catabolismpathway, gene targets for metabolic engineering of the CHO cells (CHOcell line B and parental cell line) suppressing the biosynthesis ofisovalerate, isobutyrate and 2-methylbutyrate were identified.

Results

RT qPCR analysis was performed on leucine catabolic pathway genes. Thelog expression values for the genes in the leucine pathway are shown inFIG. 33A. The gene expression data from the leucine pathway indicatethat all enzymes in the pathway were expressed at similar levels. Geneexpression for enzymes in isoleucine and valine pathway was alsoobserved to be relatively high in the parental cell line (data notshown). The data did not point towards a clear target in the BCAApathway that can explain the high rates of isovalerate, isobutyrate and2-methylbutyrate biosynthesis and secretion. However, the enzymesdownstream of the isovalerate, isobutyrate or 2-methylbutyrate nodesmight harbor a loss of function mutations, which can explain thechanneling of flux towards isovalerate, isobutyrate or 2-methylbutyrate.Since BCAT1 (or BCAT2) and BCKDHA/BCKDHB enzymes catalyzes the first twosteps in all the three branched chain amino acid pathways, knocking-downor knocking-out or inhibiting the activity of any aforementioned enzymeswas undertaken with the objective of reducing the biosynthesis ofisovalerate, isobutyrate and 2-methylbutyrate (FIG. 33B).

Example 23: Generation of Transient BCAT1 Knockdown CHO Cell Pools toSuppress the Production of BCAA Pathway Inhibitor (Isovalerate,2-Methylbutyrate and Isobutyrate) Biosynthesis

Goal

This experiment was setup to knockdown BCAT1 gene expression in CHO cellline B and CHO parental cell line in order to limit the production ofisovalerate, isobutyrate and 2-methylbutyrate in respective cellcultures.

Materials and Methods

miRNA knockdown works by expressing small RNA sequences (approximately20-25 bases) that are complimentary to a target gene sequence (e.g.BCAT1). The miRNA binds to the messenger RNA (mRNA) of the target geneforming a region of double stranded RNA. This double stranded RNA istargeted for cleavage and degradation by the cell resulting in a netdecrease in mRNA to be translated, and hence, protein to be produced.Micro RNA knockdown of BCAT1 was performed using the BLOCK-iT InduciblePol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen). FivemiRNA oligonucleotide pairs (complementary top and bottom strand ofBCAT1) were designed using an online tool called Block-iT RNAi Designerand the DNA oligonucleotides were prepared by Integrated DNAtechnologies. The oligonucleotide pairs were annealed and ligated intothe pcDNA™6.2-GW/EmGFP-miR vector. The vectors (five of them) weresequence confirmed by WyzerBiosciences (Cambridge, Mass.). The fivemiRNA vectors or a negative control vector namedpcDNA™6.2-GW/EmGFP-miR-neg (provided by Invitrogen with the kit) weretransfected into CHO parental cell line or CHO cell line B using theGenePulser XCell eletroporator (BioRad) and were left to recover for twodays. After two days, cells were either subjected to selection pressureby antibiotic (blasticidin, 10 ug/mL) for generation of stable cellpools (see Example 24), or cell lysates from the transfected cells (oruntransfected cells) were prepared in M-PER protein extraction reagent(Thermo Fisher) supplemented with cOmplete, Mini, EDTA-free ProteaseInhibitor Cocktail (Roche). The samples were analyzed by western blotusing the Novex NuPage system (Thermo Fisher). The system providespolyacrylamide gels, sample loading buffer, sample reductant, gelrunning buffer, protein mass standard, nitrocellulose membrane, andchemiluminescence detection reagent. The blots were probed with a rabbitpolyclonal anti-BCAT1 antibody (AbCam, Cat#ab110761) or a B-Actinantibody (AbCam, Cat#ab8227). The western blots were imaged using aBioRad ChemiDoc system and analyzed for levels of B-actin and BCAT1.

BCAT1 miRNA sequences used:

miRNA seq1.1a: (SEQ ID NO: 1) TGGGAGAAGCCTCACATTAAA miRNA seq2.1a:(SEQ ID NO : 2) TCTGCTGTGAGGACCACTTTG miRNA seq3.1a: (SEQ ID NO: 3)AGTGGGCACGATGAATCTGTT miRNA seq4.1a: (SEQ ID NO : 4)CACGATGAATCTGTTCCTCTA miRNA seq5.1a: (SEQ ID NO: 5)CTTGGGCAAACTGACTGATATResults

FIG. 34 shows western blot for BCAT1 and B-Actin protein levels intransiently transfected cells. B-Actin was included as a house keepinggene (or protein) and was used as an internal comparator of proteinexpression. Levels of B-Actin were similar across all the five miRNA(transient) transfections, negative miRNA control transfection anduntransfected cells (for both CHO cell line B and parental cell line).This indicated even protein loading across all the conditions (FIGS. 34Aand 34C). In CHO parental cell line transient transfections, lower levelof BCAT1 protein were observed in case of knock-down using miRNA Seq2.1a, miRNA Seq 3.1a, miRNA Seq 4.1a and miRNA Seq 5.1a. In parentalcell line transient transfections, lower level of BCAT1 protein wereobserved in all the five knock-down conditions tested. Untransfectedcontrol and the negative control showed more intense banding patternindicating higher levels of the BCAT1 proteins.

Example 24: Generation of Stable BCAT1 Knockdown CHO Cell Pools toSuppress the Production of BCAA Pathway Inhibitor (Isovalerate,2-Methylbutyrate and Isobutyrate) Biosynthesis

Goal

The main goal of this experiment was to develop stable CHO cells poolswith reduced levels of BCAT1. The objective was to further demonstratethat these stable pools had reduced capability to produce the BCAApathway inhibitor metabolites (isovalerate, 2-methylbutyrate andisobutyrate), the reduction of which resulted in better growth andproductivity.

Results

The parental and the CHO cell line B cells transfected with the fiveBCAT1 miRNA knock-down vectors were selected for stable cell pools.Stable pools were then probed for protein levels of BCAT1. All stablepools of parental cell line, generated from transfections with the fivemiRNAs, had reduced protein levels of BCAT1, to varying extent, whencompared to the stable pools generated from transfection with thenegative miRNA sequence control (FIGS. 35A and 35B). This indicatedsuccessful generation of stable cell pools with knockdown of BCAT1.Pools with lower protein levels of BCAT1 were selected and tested forproduction of inhibitors including isovalerate, 2-methylbutyrate andisobutyrate in fed-batch cultures (with and without employing HiPDOGstrategy). Cultures producing lower levels of these inhibitorymetabolites grew to higher cell densities and yielded higher titers.Equivalent knockdown of iso-enzyme BCAT2 was also performed todemonstrate reduced levels of BCAT2 production for testing for reducedproduction of inhibitors including isovalerate, 2-methylbutyrate andisobutyrate. BCAT2 is an isoenzyme form of BCAT1, which is catalyticallyand functionally equivalent to BCAT1 and performs the same function inthe leucine, isoleucine and valine pathways depicted in FIG. 33B.

TABLE 10 Abbreviations Abbreviation Description B-Actin Actin, beta MIFMacrophage migration inhibitory factor GOT1 Glutamic-oxaloacetictransaminase 1, soluble GOT2 Glutamatic-oxaloacetic transaminase 2,mitochondrial TAT Tyrosine aminotransferase FAH Fumarylacetoacetatehydrolase GSTZ1 Glutathione transferase zeta 1 (maleylacetoacetateisomerase) PAH Phenylalanine hydroxylase HPD 4-Hydroxyphenylpyruvic aciddioxygenase HGD Homogentisate 1, 2-dioxygenase DLD Dihydrolipoamidedehydrogenase IVD isovaleryl coenzyme A dehydrogenase MCCC1Methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) MCCC2Methylcrotonoyl-Coenzyme A carboxylase 2 (beta) BCKDHA Branched chainketoacid dehydrogenase E1, alpha polypeptide BCKDHB Branched chainketoacid dehydrogenase E1, beta polypeptide DBT Dihydrolipoamidebranched chain transacylase E2 BCAT1 Branched chain aminotransferase 1,cytosolic BCAT2 Branched chain aminotransferase 2, mitochondrial AUH AURNA binding protein/enoyl-coenzyme A hydratase HMGCL3-Hydroxy-3-methylglutaryl-Coenzyme A lyase ACADM Acyl-Coenzyme Adehydrogenase, medium chain GCH1 GTP cyclohydrolase 1 PTS6-Pyruvoyl-tetrahydropterin synthase SPR Sepiapterin reductase PCBD1Pterin 4 alpha carbinolamine dehydratase/dimerization cofactor ofhepatocyte nuclear factor 1 alpha (TCF1) 1 QDPR Quinoid dihydropteridinereductase BH₄ Tetrahydrobiopterin BH₄-4a- Tetrahydrobiopterin-4alpha-carbinolamine carbinolamine q-BH₂ Quinoid Dihydrobiopterin NAD⁺Oxidized nicotinamide adenine dinucleotide NADH Reduced nicotinamideadenine dinucleotide GTP Guanosine-5′-triphosphate 4x-tfxn Cell poolstransfected with four vectors containing a gene of interest and aresistance marker (PAH & geneticin or, HGD & blasticidin or, HPD &hygromycin or, PCBD1 & puromycin) 4x-control-tfxn Cell pools transfectedwith four null vectors containing only a resistance marker (geneticinor, blasticidin or, hygromycin or, puromycin) CoA Coenzyme A

REFERENCES

-   Altamirano C, Illanes A, Becerra S, Cairo J J, Godia F (2006)    Considerations on the lactate consumption by CHO cells in the    presence of galactose. Journal of Biotechnology 125: 547-556-   Bertoni J M (1981) Competitive inhibition of rat brain hexokinase by    2-deoxyglucose, glucosamine, and metrizamide. Journal of    neurochemistry 37: 1523-1528-   Clem B, Telang S, Clem A, Yalcin A, Meier J, Simmons A, Rasku M A,    Arumugam S, Dean W L, Eaton J, Lane A, Trent J O, Chesney J (2008)    Small-molecule inhibition of 6-phosphofructo-2-kinase activity    suppresses glycolytic flux and tumor growth. Molecular cancer    therapeutics 7: 110-120-   Duvel K, Yecies J L, Menon S, Raman P, Lipovsky A I, Souza A L,    Triantafellow E, Ma Q, Gorski R, Cleaver S, Vander Heiden M G,    MacKeigan J P, Finan P M, Clish C B, Murphy L O, Manning B D (2010)    Activation of a metabolic gene regulatory network downstream of mTOR    complex 1. Molecular cell 39: 171-183-   Gagnon M, Hiller G, Luan Y T, Kittredge A, DeFelice J, Drapeau    D (2011) High-end pH-controlled delivery of glucose effectively    suppresses lactate accumulation in CHO fed-batch cultures.    Biotechnology and bioengineering 108: 1328-1337-   Kim S H, Lee G M (2007a) Down-regulation of lactate dehydrogenase-A    by siRNAs for reduced lactic acid formation of Chinese hamster ovary    cells producing thrombopoietin. Applied microbiology and    biotechnology 74: 152-159-   Kim S H, Lee G M (2007b) Functional expression of human pyruvate    carboxylase for reduced lactic acid formation of Chinese hamster    ovary cells (DG44). Applied microbiology and biotechnology 76:    659-665-   Lee H L T, Boccazzi P, Gorret N, Ram R J, Sinskey A J (2004) In situ    bioprocess monitoring of Escherichia coli bioreactions using Raman    spectroscopy. Vibrational Spectroscopy 35: 131-137    Lee J S, Lee G M (2012) Rapamycin treatment inhibits CHO cell death    in a serum-free suspension culture by autophagy induction.    Biotechnology and bioengineering 109: 3093-3102-   Li B, Ryan P W, Ray B H, Leister K J, Sirimuthu N M, Ryder A    G (2010) Rapid characterization and quality control of complex cell    culture media solutions using raman spectroscopy and chemometrics.    Biotechnology and bioengineering 107: 290-301-   Morgan H P, O'Reilly F J, Wear M A, O'Neill J R, Fothergill-Gilmore    L A, Hupp T, Walkinshaw M D (2013) M2 pyruvate kinase provides a    mechanism for nutrient sensing and regulation of cell proliferation.    Proceedings of the National Academy of Sciences of the United States    of America 110: 5881-5886-   Mulukutla B C, Gramer M, Hu W S (2012) On metabolic shift to lactate    consumption in fed-batch culture of mammalian cells. Metabolic    engineering 14: 138-149-   Whelan J, Craven S, Glennon B (2012) In situ Raman spectroscopy for    simultaneous monitoring of multiple process parameters in mammalian    cell culture bioreactors. Biotechnology progress 28: 1355-1362-   Whitehouse S, Cooper R H, Randle P J (1974) Mechanism of activation    of pyruvate dehydrogenase by dichloroacetate and other halogenated    carboxylic acids. The Biochemical journal 141: 761-774-   Wlaschin K F, Hu W S (2007) Engineering cell metabolism for    high-density cell culture via manipulation of sugar transport.    Journal of biotechnology 131: 168-176-   Yi W, Clark P M, Mason D E, Keenan M C, Hill C, Goddard W A, 3rd,    Peters E C, Driggers E M, Hsieh-Wilson L C (2012)    Phosphofructokinase 1 glycosylation regulates cell growth and    metabolism. Science 337: 975-980-   Zhou M, Crawford Y, Ng D, Tung J, Pynn A F, Meier A, Yuk I H,    Vijayasankaran N, Leach K, Joly J, Snedecor B, Shen A (2011)    Decreasing lactate level and increasing antibody production in    Chinese Hamster Ovary cells (CHO) by reducing the expression of    lactate dehydrogenase and pyruvate dehydrogenase kinases. Journal of    biotechnology 153: 27-34-   Zhu G, Zhu X, Fan Q, Wan X (2011) Raman spectra of amino acids and    their aqueous solutions. Spectrochimica acta Part A, Molecular and    biomolecular spectroscopy 78: 1187-1195-   Parniak, M. A., Davis, M. D., and Kaufman, S. (1988) Effect of    alkaline pH on the activity of rat liver phenylalanine hydroxylase.    The Journal of biological chemistry 263, 1223-1230.

The invention claimed is:
 1. A cell comprising two or more exogenouswild-type genes selected from the group consisting of Pah, PCBD1, Hpd,and Hgd, wherein expression of said two or more exogenous wild-typegenes reduces the level of synthesis of growth and/or productivityinhibitors produced by the cell when compared to a cell that does notcomprise said two or more exogenous wild-type genes.
 2. The cellaccording to claim 1, wherein the cell comprises a combination ofconstructs selected from the group consisting of: (i) an expressiblenucleic acid or vector construct comprising a PCDB1 gene, (ii) anexpressible nucleic acid or vector construct comprising a Pah gene,(iii) an expressible nucleic acid or vector construct comprising a Pahgene and PCDB1 gene, (iv) an expressible nucleic acid or vectorconstruct comprising a Hpd gene, and (v) an expressible nucleic acid orvector construct comprising a Hgd gene.
 3. The cell of claim 1, whereinthe cell comprises the exogenous wild-type genes Pah and PCBD1.
 4. Thecell of claim 3, wherein the cell further comprises an exogenouswild-type gene of at least one of Hpd or Hgd.
 5. The cell of claim 3,wherein the cell is a Chinese hamster ovary (CHO) cell or a humanembryonic kidney cell (HEK)
 6. The cell of claim 1, wherein the cellcomprises the four exogenous wild-type genes Pah, PCBD1, Hpd, and Hgd.7. The cell of claim 1, wherein the cell is a Chinese hamster ovary(CHO) cell.
 8. The cell of claim 1, wherein the cell is a humanembryonic kidney cell (HEK).